RELATED APPLICATION This application claims the benefit of U.S. Provisional Application No. 61/637,185, filed Apr. 23, 2012. The entire teachings of the above application are incorporated herein by reference.
BACKGROUND Detecting, identifying, and phenotyping pathogens found in healthcare settings is critical both for diagnostic and surveillance purposes. Traditional bacterial and fungal diagnostic procedures rely on culture techniques that produce a genus or species level identification after 24-48 hours. Such tests are ordered for patients demonstrating symptoms indicative of an infection. While culture has been the standard diagnostic method for over one hundred years, its slow turnaround time means that a physician must prescribe antibiotics before knowing the identity of the organism or its drug resistances.
More recently, rapid techniques such as qPCR and mass spectrometry have allowed sub-24 hour turnaround times and enabled surveillance applications. For example, many hospitals in the United States test every patient for MRSA on admission to determine an appropriate caution level (e.g., quarantine) for patients who are at a high risk for spreading an infection to other patients. qPCR offers quick results but minimal information—a typical test only detects the presence of one or a few sequences from one organism. Testing for additional organisms or the presence of drug resistance or virulence genes adds substantially to the cost of the test.
A test that offers sub-24 hour turnaround time while identifying a large number of organisms would offer many benefits in a healthcare setting including broad-range surveillance and faster prescriptions of the most appropriate antibiotic. The present application discloses compositions, kits, and methods that can be used to detect any or several of a large set of organisms present in a sample as well as a number of families of drug resistance genes.
SUMMARY Provided herein are compositions, kits, and methods for identifying an organism. The organism can be a microbe, microorganism, or pathogen, such as a virus, bacterium, or fungus. In one embodiment, an organism is distinguished from another organism. In another embodiment, a strain, variant or subtype of the organism is distinguished from another strain, variant, or subtype of the same organism. For example, a strain, variant or subtype of a virus can be distinguished from another strain, variant or subtype of the same virus.
In some aspects, a probe set for identifying pathogenic organisms or strains in a sample comprising a plurality of probes that, when implemented in an assay, allows for detecting and distinguishing at least 5 different strains, variants, or subtypes of at least 3 pathogenic organisms, wherein each probe in said plurality comprises a first sequence that hybridizes to a 5′ end of a target sequence of said pathogen, a 3′ end of said pathogen, or to said target sequence is provided.
In some embodiments, pathogen strains or organisms comprise a virus, bacterium, or fungus. In some embodiments, the at least 3 pathogenic organisms include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Acinetobacter baumanii, Clostridium difficile, Escherichia coli, Enterobacter (aerogenes, cloacae, asburiae), Enterococcus (faecium, faecalis), Klebsiella pneumoniae, Proteus mirabilis, Candida albicans, and Pseudomonas aeruginosa; or subtypes or strains thereof.
In some embodiments, the probe set can not only detect and distinguish between the at least 3 organisms but can also distinguish between common strains or subtypes of the organisms. In some embodiments, the probe set detects and distinguishes among the organisms responsible for more than 90% of the hospital acquired infections at some site.
In one aspect, a probe set for identifying the presence of drug resistance genes in the organisms in a sample comprising a plurality of probes that, when implemented in an assay, allows for detecting and distinguishing at least 3 classes of resistance genes, wherein each probe in said plurality comprises a first sequence that hybridizes to a 5′ end of a target sequence of said pathogen, a 3′ end of said pathogen, or to said target sequence is provided.
In one aspect, a kit containing any probe set described herein and the reagents and protocol to capture the target sequences of the organisms present in the input sample is provided.
In some aspects, a kit for the simultaneous detection of pathogens including three or more of the organisms listed in Table 2 is provided. In some embodiments, the kit is for research use. In some embodiments, the kit is a diagnostic kit. In some aspects, a kit for the simultaneous detection of antibiotic resistance genes including three or more of the genes listed in Table 3 is provided. In some embodiments, the kits described herein can be used to prepare DNA for massively parallel sequencing. In some embodiments, the kits described herein can provide molecular barcodes for the labeling of individual samples. In some embodiments, the kits described herein can include at least 10 of the probe sequences listed in Table 1.
In some embodiments, the kits described herein can be used to circularize single-stranded DNA probes by: (i) hybridization to a complementary target DNA sequence, (ii) extension across a gap by DNA polymerase, and (iii) ligation of the extended probe to form a single stranded, covalently closed circular DNA molecule.
In one aspect, a composition comprises a probe set for identifying pathogenic organisms or strains in a sample comprising a plurality of probes that, when implemented in an assay, allows for detecting and distinguishing three or more of the organisms listed in Table 2, wherein each probe in said plurality comprises a first sequence that hybridizes to a 5′ end of a target sequence of said pathogen, a 3′ end of said pathogen, or to said target sequence. In one embodiment, the plurality of probes, when implemented into an assay, allows for the substantially simultaneous detection and distinguishing of three or more of the antibiotic resistance genes listed in Table 3 is provided.
In one aspect, a composition comprises a probe set for identifying antibiotic resistance genes of pathogenic organisms or strains in a sample comprising a plurality of probes that, when implemented in an assay, allows for detecting and distinguishing three or more of the antibiotic resistance genes listed in Table 3, wherein each probe in said plurality comprises a first sequence that hybridizes to a 5′ end of a target sequence of said pathogen, a 3′ end of said pathogen, or to said target sequence is provided.
In one aspect, a composition comprises a probe set for identifying pathogenic organisms or strains in a sample comprising a plurality of probes that, when implemented in an assay, allows for detecting and distinguishing three or more organisms that cause Hospital Associated Infections (HAIs) at some site, wherein each probe in said plurality comprises a first sequence that hybridizes to a 5′ end of a target sequence of said pathogen, a 3′ end of said pathogen, or to said target sequence is provided. In some embodiments, the three or more organisms that cause HAIs at some site comprise organisms responsible for more than 90% of the hospital acquired infections at some site. In some embodiments, the three or more organisms that cause HAIs at some site comprise organisms responsible for more than 60% of the hospital acquired infections at some site. In some embodiments, the three or more organisms that cause HAIs at some site comprise organisms responsible for more than 30% of the hospital acquired infections at some site. In some embodiments, the site is a surgical site, catheter, ventilator, intravenous needle, respiratory tract catheter, medical device, blood, blood culture, urine, stool, fomite, wound, sputum, pure bacterial culture, mixed bacterial culture, bacterial colony, or any combination thereof.
In some embodiments, a probe set is operable to detect CARB, CMY, CTX-M, GES, IMP, KPC, NDM, ampC, OXA, PER, SHV, VEB, VIM, ermA, vanA, canB, mecA, or mexA family or classes of genes, or any combination thereof. In some embodiments, some of the genomic regions chosen as target sequences are known to be highly conserved such that each genus or species tends to contain a single version of the region, thus allowing genus or species identification. In some embodiments, some of the genomic regions chosen as target sequences are known to be highly variable such that each strain or substrain will contain a different version of the region, thus enabling strain or substrain identification and differentiation. In some embodiments, some portion of a plurality of the selected target sequences are sequenced simultaneously and then mapped to a database of reference sequences to determine the most likely identities of the organisms or genes present in the sample. In some embodiments, some portion of a plurality of the selected target sequences are sequenced simultaneously and then assembled into one or more consensus sequences. When sequencing information is gathered from the probes for antibiotic resistance genes, for plasmids, and for an organism, a distinguishing fingerprint can be derived for the pathogen, and can serve as means to identify the source and extent of an outbreak.
In one aspect, a kit comprising one or more reagents, wherein the reagents comprise a probe set according to claims 1-11, reagents for obtaining a sample, reagents for extracting nucleotides from a sample, enzymes, reagents for amplifying a region of interest, reagents for purifying nucleotides, reagents for purifying captured regions of interest, buffers, sequencing reagents, or any combination thereof, wherein the reagents allow for the capture of target sequences of three more pathogens listed in Table 2 is provided.
In one aspect, a kit comprising one or more reagents, wherein the reagents comprise a probe set according to claims 1-11, reagents for obtaining a sample, reagents for extracting nucleotides from a sample, enzymes, reagents for amplifying a region of interest, reagents for purifying nucleotides, reagents for purifying captured regions of interest, buffers, sequencing reagents, or any combination thereof, wherein the reagents allow for the capture of target sequences of three or more antibiotic resistance genes listed in Table 3 is provided.
In one aspect, a kit comprising one or more reagents, wherein the reagents comprise a probe set according to claims 1-11, reagents for obtaining a sample, reagents for extracting nucleotides from a sample, enzymes, reagents for amplifying a region of interest, reagents for purifying nucleotides, reagents for purifying captured regions of interest, buffers, sequencing reagents, protocol or any combination thereof, wherein the reagents allow for the capture of target sequences of three or more pathogens listed in Table 2 and capture of target sequences of three or more antibiotic resistance genes listed in Table 3 is provided.
In some embodiments, the reagents allow the capture reaction to be performed in a single tube. In some embodiments, the reagents allow the capture reaction to be performed in less than three hours. In some embodiments, the reagents allow the capture reaction to be performed in less than two hours. In some embodiments, the detection of the three or more pathogens occurs substantially simultaneously.
In some embodiments, the plurality of probes comprises at least 3 of the probe sequences listed in Table 1. In some embodiments, each probe comprises the first sequence that hybridizes to a 5′ end of said target sequence and a second sequence that hybridizes to a 3′ end of said target sequence. In some embodiments, the probe set can distinguish between strains or subtypes of the organisms. In some embodiments, the detection the three or more antibiotic resistance genes occurs substantially simultaneously. In some embodiments, the detection of the three or more pathogens and the three or more antibiotic resistance genes occurs substantially simultaneously.
In some embodiments, a kit allows for preparation of DNA for massively parallel sequencing. In some embodiments, a kit further comprises molecular barcodes for the labeling of individual samples.
In some embodiments, the probe set of a kit comprises at least 10 of the probe sequences listed in Table 1. In some embodiments, the probe set of a kit comprises at least 20 of the probe sequences listed in Table 1.
In some embodiments, kit reagents can be used to circularize single-stranded DNA probes by: (i) hybridization to a complementary target DNA sequence, (ii) extension across a gap by a DNA polymerase, and (iii) ligation of the extended probe to form a single stranded, covalently closed circular DNA molecule.
In one aspect, a method of identifying an organism or pathogenic strain, variant or subtype comprising: a) contacting a sample with a plurality of probes listed in Table 1, wherein said plurality of probes detects and distinguishes at least 3 different organisms or pathogenic strains listed in Table 2, or variants or subtypes thereof; b) hybridizing a 5′ end of a target sequence of said organisms or pathogenic strains, or variants or subtypes thereof, a 3′ end of said target sequence, or said target sequence with a probe of said plurality; c) sequencing said target sequence; and d) identifying from said sequencing said organisms or pathogenic strains, or variants or subtypes thereof is provided.
In one embodiment, the method is performed in less than 12 hours. In one embodiment, the identifying is performed in less than 3 hours. In one embodiment, the identifying is performed in less than 2 hours. In one embodiment, the identifying is with at least 99% specificity or sensitivity.
In one aspect, a method of stratifying a host into a therapeutic group comprising: a) contacting a sample from said host with a plurality of probes listed in Table 1, wherein each probe specifically distinguishes different non-host organisms or pathogenic strains listed in Table 2, or variants or subtypes thereof; b) hybridizing a 5′ end of a target sequence of a non-host organism or pathogen, a 3′ end of said target sequence, or said target sequence with a probe of said plurality; c) sequencing said target sequence; d) determining an identity of said non-host organism or pathogenic strain, or variant or subtype thereof, from said sequencing; and e) stratifying said host into a therapeutic group based on said identity is provided. In one embodiment, the method further comprises determining the genotype of the host from the sample.
In some embodiments, an additional non-host organism is identified. In some embodiments, an additional strain, variant or subtype of said organism or pathogen is identified. In some embodiments, the therapeutic group differs than a therapeutic group in which only one of the non-host organisms is identified. In some embodiments, the therapeutic group differs than a therapeutic group in which only one of said strains, variants, or subtypes of said pathogen is identified.
INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
FIG. 1 depicts an exemplary kit configuration, indicating the position of samples and barcoding reagents within the supplied materials within the kit.
FIG. 2 provides a matrix depiction of a subset of a probeset for discrimination of genus and species amongst many genomes of various organisms. Each column on the x-axis indicated a single probe capture region, and each row indicates a reference database genome within the genus or species labeled. Dark boxes indicate that a probe is not predicted to provide sequence for this organism, whereas white boxes indicate that this probe is predicted to bind and provide sequence enabling the detection of this organism.
FIGS. 3A-3B depict exemplary plots of data that can be used to quantify target organisms including (FIG. 3A) Acinetobacter and (FIG. 3B) S. saprophyticus. In each case, genomic DNA isolated from a culture of each organism was quantified and a dilution series of 4 orders of magnitude aliquoted. Each aliquot was sequenced in triplicate and the total sequencing reads per aliquot divided by the number of internal control reads to produce a normalized quantitation of the DNA present in the sample. The plotted results indicate a highly linear and quantitative relationship between sequenced reads detected and input DNA.
FIG. 4 depicts a graph showing that the kits described herein can resolve mixed samples containing multiple organisms. In each case, genomic DNA isolate from a culture of each organism was quantified and a aliquoted into a sample at even copy numbers, with the sample matrix indicating mixes of up to 5 distinct genomes within each sample. Each sample was sequenced in duplicate and the total sequencing reads for each individual genome per sample divided by the number of internal control reads to produce a normalized relative quantitation of the each of the genomic DNA species present in the sample. The graphed results indicate accurate detection of multiple species within a mixed DNA sample.
FIG. 5 depicts a plot demonstrating strong correlation (R2=0.98) between the log normalized counts obtained via PGM vs log normalized counts obtained via qPCR. Genomic DNA from an organism was quantified and a aliquoted as a dilution series over ˜5 orders of magnitude. Each sample was sequenced in triplicate and the total sequencing reads for the genome per sample divided by the number of internal control reads to produce a normalized relative quantitation genomic DNA species present in each aliquot. qPCR was also performed in triplicate on each sample using a genome specific primer pair, and the qPCR relative copy number then plotted against the sequencing data (PGM normalized count). The results demonstrate a linear agreement with quantitation by qPCR over >4 orders of magnitude.
FIG. 6 depicts a plot of the ratio of viral (HIV) reads to GFP against the initial template concentration in the reaction. cDNA from HIV was quantified and a aliquoted as a dilution series over ˜4 orders of magnitude. Samples were prepared in the presence of 1000 genome equivalents of human DNA isolated from cultured HEK-293 cells, or in the absence of background competing DNA. Each sample was sequenced and the total sequencing reads for the genome per sample divided by the number of GFP internal control reads to produce a normalized relative quantitation of HIV cDNA present in each aliquot. No significant difference was observed in the number of sequencing reads per sample in the presence or absence of competing background Human DNA.
FIG. 7 depicts a plot comparing the detection of cDNA from 2 HIV strains (CN009 and CN006) obtained via PGM vs. MiSeq. The plot is shown as the adjusted GFP read count against the CN009 Template count. In each case, cDNA from HIV CN009 was quantified and a aliquoted as a dilution series over ˜5 orders of magnitude. Into each CN009 aliquot, 3000 genome equivalents of CN006 genome were also added. Each sample was sequenced in duplicate and the total sequencing reads for each individual genome per sample divided by the number of internal control reads to produce a normalized relative quantitation of the each of the genomic DNA species present in the sample. The plots indicated a consistent level of CN006 detection detected per sample, and a linear detection of CN009 over >4 orders of magnitude. This also demonstrates the detection of two species at minor variant frequencies of as low as 1%.
FIG. 8 depicts a plot of sequencing counts per probe within a probeset, for replicate sample B1 against replicate sample B2. The plot demonstrates a highly linear and reproducible probeset internal performance.
FIGS. 9A-9B depict plots of the ratio of minor:major pathogen PGM reads against the percent ratio of minor:major pathogen in the reaction for (FIG. 9A) minor pathogen detected to 1% major pathogen (S. epidermidis and E. coli) and (FIG. 9B) minor pathogen detected to 10% major pathogen (S. saprophyticus and A. baumannii). In each case, genomic DNA isolated from a culture of two organisms was quantified and aliquoted into a sample at a ratio of 10:1, 1:1, 1:10 and 1:100. Each sample was sequenced in triplicate and the total sequencing reads for each individual genome per sample divided by the number of internal control reads to produce a normalized relative quantitation of the each of the genomic DNA species present in the sample. The graphed results indicate accurate detection of multiple species within a mixed DNA sample down to a minor variant level of at least 1%.
FIG. 10A describes a list of assay components within the HAI BioDetection kit.
FIG. 10B illustrates the layout of customer samples and control samples of two 8 well strips that the BioDetection assay is performed within.
FIG. 10C indicates validated performance specifications and criteria for the HAI BioDetection kit.
FIG. 11A describes the multilevel structure of the HAI BioDetection probeset.
FIG. 11B provides a matrix depiction of two probes within the BioDetection probset to illustrate discrimination of species and strain amongst many genomes of Staphlyococcus. Each column on the x-axis indicates a SNP detected by either probe 1 or probe 2 capture region, and each row indicates a reference database genome within the genus or species labeled. Black boxes indicate that a probe is not predicted to provide sequence for this organism, whereas shaded boxes indicate that this probe is predicted to bind and provide sequence enabling the detection of this organism.
FIG. 11C describes the levels of multiplexity achieved by the HAI BioDetection kit by assaying many sequence variants within a sample, compared to the single nucleotide discriminatory ability of a PCR primer.
FIG. 12 illustrates the workflow from input sample, either a purified genomic DNA from culture, e.g. DNA enriched from a swab of a patient wound site. The BioDetection kit workflow is illustrated in elapsed time (from t=12.00) and the workflow timing for each individual step is broken out to the left of the workflow. A barcoding primer set of allows 16 to 96 samples to be sequenced simultaneously in one run on a sequencing platform (in this illustration and Ion Torrent PGM, but alternatively and Illumina MiSeq or HiSeq platform). The interpretation box illustrates a computational software and graphical display of simplified data output.
FIG. 13 illustrates a graphical display of summarized sequencing results from the BioDetection kit. The graphical display is subdivided into Genus, species and strain level detection results, resistance gene information if resistance loci are detected, the readcount for samples and internal controls, and also any potential warnings due to poor sample performance. A color-coded similarity score (green=similar, yellow=moderate similarity, red=little similarity) and a similarity score absolute value, are calculated for the sequenced similarity detected by the kit and compared to the next most related organism at genus, species and strain level using a reference database of published genomic sequences, and containing previous genome sequences detected by the BioDetection kit. In the illustrated example, the sample has been demonstrated to contain both Enterococcus faecalis as the primary species, and Escherichia coli a minor species present within the sample. The samples are 65.4% and 74% homologous to the nearest neighbor strains described.
FIG. 14 illustrates a schematic comparison of the turnaround time and workflow steps to generate substrain level resolution and drug resistance typing of bacterial samples using either traditional microbiology, and combination of PCR and/or Mass spectroscopy, whole genome sequencing (WGS) or the BioDetection kit. A clear advantage is illustrated with the BioDetection kit in terms of fewer workflow steps and faster achievement of substrain resolution and drug resistance typing compared to these alternative methods.
FIG. 15A describes a collection of 38 MRSA samples were subtyped using both HAI BioDetection Kit and spa locus VNTR typing (using PCR and Sanger sequencing). Sequence regions captured using the BioDetection kit were used to construct a phylogenetic tree was constructed using sequence data, and each sample was annotated with spa-typing result for the same sample. The tree demonstrates the discrimination of samples with the same spa-type into multiple unique isolates using the BioDetection kit. Further the grouping clustering generated by the BioDetection kit largely groups according to spa-type, as would be predicted for more closely related samples.
FIG. 15B describes the number of sequence variants detected amongst 38 sequenced with the BioDetection kit, or typed by spa type, or 8 representative samples encapsulating the broad phylogenetic tree structure and then Sanger sequenced using then MLST subtyping amplicons, or the 16S ribosomal sequencing amplicon. The total number of MRSA samples uniquely discriminated by each approach is also described.
FIG. 15C describes 4 bacterial cohorts sequenced using the BioDetection kit. The table indicates the number of samples per cohort, and the number of % of the samples that were discriminated into unique isolates. The data demonstrates that the HAI kit is capable of near unique discrimination of bacterial isolates within many large cohorts.
FIG. 16A is an in silico model predicting the superiority of the present invention over VNTR approaches. Thirty-three genome sequences were extracted from references databases for which whole genome sequence assemblies were available. An in silico analysis extracted regions of these genomes that are assayed using MLVA, MLST and spa subtyping methods. The discriminatory index (defined as the % of total genomes discriminated into unique isolates) of each technique was calculated based upon the assayed regions, and compared with the regions assayed by the BioDetection kit. The BioDetection kit discriminated all samples into unique groups, and demonstrated a higher discriminatory index than other assays.
FIG. 16B is a tangelgram demonstrating better phylogeny reproduction by the BioDetection kit. The sequences extracted by the in silico analysis of Figure x2 were used to construct phylogenetic trees to describe the relationships between samples. The whole genome sequence (WGS) was used as a reference tree, and the BioDetection and MLVA constructed trees compared using a tangelgram figure. Red and pink lines represent regions of the tree that are significantly different between methods, whereas parallel grey lines illustrate relationships that are described equivalently by both methods. The figure demonstrates significant discordance between the MLVA and WGS trees, but largely comparable phylogenetic relationships described by the WGS and BioDetection trees. This data indicates that the evolutionary relationships described by the BioDetection kit are a more accurate appraisal of the whole genome relationships and evolutionary distance between samples than MLVA approaches. Similar significant discordances were observed to between WGS and spa-typing and MLST trees compared to MLVA.
FIG. 17 demonstrates detection from DNA isolated from stool, urine and sputum. Sputum, stool and urine sample derived from human individuals were spiked with genomic DNA isolated from cultured bacteria. Samples were extracted using standard DNA extraction methods, such that each sample contained some amount of gDNA plus any additional complex biomolecules that are carried through the extraction from these sample types (heme, complex polysaccharides and potential enzyme inhibitors). Each isolated DNA sample was assayed using the BioDetection kit and results are tabulated. This data demonstrates accurate species and strain detection from DNA isolated from sputum, urine and stool samples, plus identification of resistance genes present within each sample.
FIG. 18 is a summary of 707 bacterial samples sequenced and identified using the BioDetection kit. The table demonstrates the capability of the kit to detect species not listed and validated within the performance specifications, due to the broad detection and discriminatory ability of the selected sequence capture regions.
FIG. 19 shows detection of VRE from rectal swabs. A collection of n=24 positive and negative rectal swab samples screened by microbial culture were collected after primary screening at a hospital laboratory. Bound DNA released into a PBS wash solution by incubation for 1 hr at 37 degrees centrigrade, isolated using a gDNA extraction kit and then assayed using the BioDetection kit. The resulting data was tabulated to indicate the detection of organisms and drug resistance genes (plus read counts) from each sample, and comparison to the clinical surveillance by culture. This data demonstrates accurate species, strain and resistance gene detection from rectal swabs. In particular the data illustrates detection of multiple Enterococcus strains, plus vancomycin resistance, and additional co-present species on the rectal swabs, such as E. coli and K. pneumoniae. Further, sample PGCA963 demonstrates low level E. faecium and E. coli detection in a culture negative sample.
FIG. 20 describes a summary of drug resistance loci detected over n=707 clinical isolates and clinical specimens sequenced using the HAI kit. Read count for each marker exceeds a minimum of 10 reads and often incorporates detection of multiple sequences within the gene, providing high confidence detection. This table demonstrates a range of drug resistance markers confirmed for detection by the BioDetection kit.
FIG. 21 demonstrates a comparison of 2 samples sequenced using the BioDetection kit from clinical Klebsiella isolates. The sequence represents the captured sequence of a single probe within the BioDetection probeset. The pairwise sequence comparison illustrates mismatches between a single probe loci (of multiple discriminating probes) and indicates that even a single loci commonly contains multiple SNPs of high confidence discrimination between closely related species.
FIG. 22A shows high confidence SNP calling by readcount vs WGS. A cohort of 20 MRSA samples were sequenced using the BioDetection kit on an Ion Torrent PGM, and a Nextera™ whole genome sequencing approach on a MiSeq. Samples were sequenced on a Ion 316 chip (˜3.2M reads), and a single MiSeq run (˜15M reads). Reads were aligned to a reference genome and coverage compared between sequencing approaches. The plots describe the genomic coordinates (x-axis) and the log 10 sequencing read depth at each nucleotide (y-axis) for 3 individual probes. The BioDetection kit generates considerably higher readcounts (10-100 fold) at discriminatory regions between samples, enabling higher confidence SNP calling for this targeted sequencing vs the low read depth of whole genome sequencing. This also supports accurate detections for each of the SNPs by independent library constructions using different sequencing technologies.
FIG. 22B shows genomic coordinates. Two sequence alignments compare the consensus read sequence at 2 regions captured by both HAI BioDetection kit, and Nextera Nextera™ whole genome sequencing and reference genome alignment. For samples TC14, TC5 and TC4, the sequences show agreement for detection of an indel within sample TC14, and two SNPs within TC14 relative to TC4 and TC5.
DETAILED DESCRIPTION Approximately one out of every twenty hospitalized patients will contract a nosocomial infection, more commonly known as a hospital-acquired infection (HAI). More than 70 percent of the bacteria that cause HAIs can be resistant to at least one of the antibiotics most commonly used to treat them. Early detection can be important for controlling the spread of hospital-acquired infections. After culturing for growth and isolation of pathogens, clinical microbiology laboratories may rely on observable phenotype and simple biochemical assays to determine the bacterial type and antibiotic sensitivity. Determining the most effective antibiotic treatment for the infected patient, not the causal agent of the infection, is usually the prerogative of the physician. The resolution of conventional microbiological assays may be insufficient to determine the precise genotype underlying antibiotic resistance. Consequently, the same organism can infect multiple patients, and the spread of infection can go unnoticed for long periods.
Urinary tract infection (UTI) is the most common hospital-acquired infection. UTIs account for about 40 percent of hospital-acquired infections, and an estimated 80 percent of UTIs are associated with urinary catheters. Pneumonia is the second most common HAI. In critically ill patients, ventilator-associated pneumonia (VAP) is the most common nosocomial infection. VAP can double the risk of death, significantly increase intensive care unit (ICU) length of stay, and can add to each affected patient's hospital costs.
A key problem for microbiology labs is the turnaround time from receiving a microbial sample to determining key actionable information for patient care, such as antibiotic drug resistance within the sample, or strain identification for comparison to known high-risk strains. Existing technologies such as PCR or mass spectroscopy have allowed the turnaround time to be improved relative to classical methods for some actionable information, such as species identification, or presence of a select few drug resistance genes, but there are few practical approaches to assaying the large number of drug resistance genes or key species needed to be identified to confidently predict patient treatment.
DNA microarray offers broad detection ability for genomic loci, but is complicated by slow sample preparation and false positive and false negative sample results due to the hybridization based approach. Targeted DNA sequencing using the BioDetection kit allows the greater breadth of target detection, and higher resolution and higher accuracy discrimination due to the single base accuracy of DNA sequencing.
A second competing approach to targeted sequencing is whole genome sequencing. This approach has several disadvantages relative to the targeted sequencing approach provided by the invention. First, whole genome libraries contain many uninformative regions that are identical between the majority of isolates in a species, and thus provide no information to discriminate. These worthless reads mean that many more WGS reads are required per sample to capture informative regions, and prevent higher numbers of samples to be multiplexed into a single sequencing channel to amortize sequencing costs. Second, WGS libraries contain a representative fraction of any DNA present within a sample. As such, primary samples containing human tissue, or many uninteresting bacteria from the perspective of patient health, will comprise mainly of unwanted human or commensal bacterial reads. Efficient detection of important bacteria and drug resistance genes within a sample requires a more efficient targeted approach. Thirdly, library preparation times are slower and more laborious using WGS approaches, and the data analysis time significantly longer than that of a targeted sequencing approach in which only key informative regions are analyzed. This faster analysis reduces turnaround time and costs, and allow simplified data representations for easier understanding for clinical scientists unfamiliar with next generation sequencing data.
Provided herein are compositions, methods, systems and kits for detecting an organism, such as a pathogen, such as a pathogen that causes HAIs, as well as methods for using the system to identifying and detect the organism. The system can comprise a probe or plurality of probes. Also provided herein, are compositions, methods, systems and kits for detecting an organism, such as a pathogen, such as a pathogen that causes HAIs, and detecting and identifying antibiotic resistance genes, which, in some embodiments, can be performed simultaneously.
Probes In some embodiments, the invention provides panels of probes and methods of using them, where the panels include circularizing capture probes, such as molecular inversion probes. Basic design principles for circularizing probes, such as simple molecular inversion probes (MIPs) as well as related capture probes are known in the art and described in, for example: Nilsson et al., Science, 265:2085-88 (1994); Hardenbol et al., Genome Res.; 15:269-75 (2005); Akharas et al., PLOS One, 9:e915 (2007); Porecca et al., Nature Methods, 4:931-36 (2007); Deng et al., Nat. Biotechnol., 27(4):353-60 (2009); U.S. Pat. Nos. 7,700,323 and 6,858,412; and International Publications WO 2011/156795, WO/1999/049079 and WO/1995/022623, all of which are incorporated by reference in their entirety.
A system for detection of an organism, such as identifying a strain, variant or subtype of a pathogen, can comprise a mixture or probe set comprising a plurality of probes. The target organism for a particular probe may be any organism, such as a viral, bacterial, fungal, archaeal, or eukaryotic, organisms, including single cellular and multicellular eukaryotes. In particular embodiments, a target organism is a pathogen. In some embodiments, target organisms include organisms associated with or that cause HAIs, such as those organisms provided in Table 2.
In some embodiments, each single-stranded capture probe can hybridize to two complementary regions on a target DNA with a gap region in between. An enzyme, such as DNA polymerase, can be used to fill in the gap using the target as template, and stop adding nucleotides when it reaches the phosphorylated 5′-terminus of the hybridized probe. An enzyme, such as a thermostable ligase, can be used to covalently close the extended probe to form a circular molecule. Exonucleases can be used to digest away residual probe molecules. The filled-in, circularized probe can be resistant to exonuclease digestion, and can serve as template for preparation of the sequencing library by known methods, such as PCR. Sample-associated barcodes can be added and can enable multiple barcoded samples to be blended and analyzed together, such as on a DNA sequencer.
A probe can refer to a sequence that hybridizes to another sequence. The probe can be a linear, unbranched polynucleic acid. The probe can comprise two homologous probe sequences separated by a backbone sequence, where the first homologous probe sequence is at a first terminus of the nucleic acid and the second homologous probe sequence is at the second terminus to the nucleic acid, and where the probe is capable of circularizing capture of a region of interest of at least 2 nucleotides. Circularizing capture can refer to a probe becoming circularized by incorporating the sequence complementary to a region of interest.
In a preferred embodiment, the probes contain two arms, joined by a backbone, that hybridize to a target sequence. A polymerase molecule can extend the 3′ end of the probe by copying a target region into a probe molecule. A ligase molecule can circularize a probe molecule by joining the 3′ end of the copied target to the 5′ end of the original probe molecule.
In one embodiment, probe arms can hybridize to the target nucleic acid molecule, surrounding the capture region; a polymerase extension can fill in the gap between the arms and a ligase can create a circular molecule out of the extended probe. After an exonuclease digestion removes the original template molecules, primers can be used to amplify the captured probes. The primers can contain a 3′ end homologous to the backbone (forward) and its reverse complement (reverse primer). The 5′ of the primer may contain a sequencing adapter for a particular next generation sequencing platform and may also contain a barcode sequence between the 5′ and 3′ segments such that multiple samples, each amplified with primers containing a sample-specific barcode, can be multiplexed into a single sequencing run. As the two probe arms are linked by a backbone, on-target binding is energetically favorable, even when many (hundreds, thousands, or tens of thousands) of probes are present in a single reaction (compare to PCR, in which one primer of a pair may hybridize and extend at an off-target locus). As with PCR, each MIP can capture a well-defined region of the target sequence (compare to hybridization capture methods, which yield a variety of molecules centered around the target).
In a preferred embodiment, a backbone of a probe molecule contains the same sequence in all probes. A backbone can contain two primer binding sites that allow amplification of probe arms and a captured target sequence. In a preferred embodiment, the primers used may contain a barcode to allow multiple samples to be separated after simultaneous sequencing. In a preferred embodiment, the primers also contain 5′ ends that adapters for a next-generation sequencing platform such as the Ion Torrent PGM, Illumina MiSeq, Illumina HiSeq, Nanopore, etc (FIG. 1).
The probe set can include large number of probes, e.g., 10, 20, 30, 40, 50, 100, 200, 400, 500, 1000, 2000, 3000, 4000, 5000, 10000, 20000, 40000, 80000, or more. The probe set can include one or more probes directed to a large number of different target organisms, e.g., at least 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1250, 1500, 1750, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more different target organisms. In some embodiments, a mixture including one or more probes to a plurality of target organisms contains only one probe to a target organism. In other embodiments, the mixture contains more than one probe to a target organism, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 probes for a target organism. In certain embodiments, such as embodiments designed for use with patient test samples, the mixture further includes probes with homologous probe sequences that specifically hybridize to the host genome for applications such as host genotyping. In some embodiments, the mixtures of the invention further comprise sample internal calibration standards.
In one embodiment, the plurality of probes can detect at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, or 2000 different organisms or pathogens. In another embodiment, the plurality of probes can detect at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1250, 1500, 1750, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more different strains, variants or sub-types of a pathogen or different strains or sub-types of different pathogens. In one embodiment, the probe set identifies detects at least 2 different bacterial or fungal strains. In another embodiment, the probe set identifies at least 50 different organisms, such as 50 different pathogens, or 50 different strains or subtypes of a pathogen, such as Staphylococcus aureus.
In another embodiment, the probe set can comprise probes capable of detecting a single molecule of a pathogen, thereby detecting, distinguishing or identifying the pathogen.
Each probe in the probe set can comprise the same or different backbone size, sequence, chemistries, configuration of barcodes and sequences, specific sequences for probe enrichment, target sites for probe cleavage, hybridization arm physical and chemical properties, probe identification regions, low structure optimized design, or any combination thereof. A probe may be selected to screen key loci for pathogenicity and/or drug susceptibility, and a genetic fingerprint or genotype for each sub-strain that contains key phenotypic information is generated.
In another embodiment, the probe comprises a first sequence that hybridizes to a 5′ end of a target sequence and a second sequence that hybridizes to a 3′ end of a target sequence, wherein the target sequence can be used to identify, detect, or distinguish an organism, such as pathogen. In some embodiments, the probes in the mixture each comprise a first and second homologous probe sequence—separated by a backbone sequence—that specifically hybridize to a first and second sequence (such as sequences 3′ and/or 5′ to a target sequence, respectively) in the genome of at least one target organism. In some embodiments the first and second homologous probe sequences are not complementary to the target sequence, but ligate to the 5′ and 3′ termini of a target nucleic acid, e.g. a microRNA, and possess appropriate chemical groups for compatibility with a nucleic acid-ligating enzyme, such as phosphorylated or adenylated 5′ termini, and free 3′ hydroxyl groups. The probe can be capable of circularizing capture of a region of interest.
In some embodiments, the homologous probe sequences or the sequences of the probe that hybridize or are homologous to the 3′ and/or 5′ region of a target sequence specifically hybridizes to target sequences in the genome of their respective target organism, but do not specifically hybridize to any sequence in the genome of a predetermined set of sequenced organisms—the exclusion set. In embodiments related to probes that do not hybridize directly to the capture target, the ‘homologous probe sequences’ are designed specifically to not substantially hybridize to any sequence within a defined set of genomes, i.e., an exclusion set. In the case of biological samples from a subject, the exclusion set includes the host's genome. In particular embodiments, the exclusion set also includes a plurality of viral, eukaryotic, prokaryotic, and archaeal genomes. In more particular embodiments, the plurality of viral, eukaryotic, prokaryotic, and archaeal genomes in the exclusion set may comprise sequenced genomes from commensal, non-virulent, or nonpathogenic organisms. In still more particular embodiments, the exclusion set for all probes in a mixture share a common subset of sequenced genomes comprising, for example, a host genome and commensal, non-virulent, or non-pathogenic organisms. In general, the exclusion set varies between probes in the mixture so that each probe in the mixture does not specifically hybridize with the target sequence of any other probe in the mixture.
In some embodiments, the sequences 3′ and/or 5′ to a target sequence are separated by a region of interest (e.g., the target sequence) of at least two nucleotides. In particular embodiments, they are separated by at least 5, 6, 7, 8, 9, 10, 12, 14, 18, 20, 25, 30, 50, 75, 100, 150, 200, 300, 400, 600, 1200, 1500, 2500, or more nucleotides. In some embodiments, the first and second target sequences are separated by no more than 5, 6, 7, 8, 9, 10, 12, 14, 18, 20, 25, 30, 50, 75, 100, 150, 200, 300, 400, 600, 1200, 1500, or 2500 nucleotides.
In some embodiments, probes can be designed to capture conserved regions, and upon DNA sequencing, can reveal polymorphisms and genetic aberrations that allow for the resolution of known or novel variants or closely related strains of organisms. In some embodiments two or more probes can be used for one or more or every organism wished to be tested for, which can permit discrimination of closely related organisms, even when a sample comprises more than one organism.
In one aspect, the probes in the probe set each comprising homologous probe sequences which are substantially free of secondary structure, do not contain long strings of a single nucleotide (e.g., they have fewer than 7, 6, 5, 4, 3, or 2 consecutive identical bases), are at least about 8 bases (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 27, 28, 30, or 32 bases in length), and have a Tm in the range of 50-72° C. (e.g., about 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62° C.). In some embodiments the first and second homologous probe sequences are about the same length and have the same Tm. In other embodiments, length and Tm of the first and second homologous probe sequences differ. The homologous probe sequences in each probe may also be selected to occur below a certain threshold number of times in the target organism's genome (e.g., fewer than 20, 10, 5, 4, 3, or 2 times).
The backbone sequence of the probes may include a detectable moiety and a primer-binding sequence. In some embodiments, the backbone sequence of the probes comprises a second primer. In particular embodiments, the detectable moiety is a barcode. In certain embodiments the backbone further comprises a cleavage site, such as a restriction endonuclease recognition sequence. In certain embodiments, the backbone contains non-WatsonCrick nucleotides, including, for example, abasic furan moieties, and the like.
In another aspect, the invention provides a kit comprising one or more sets of probes, such as one or more sets of probes from the probes provided in Table 1. In one embodiment, a kit comprises one or more reagents for obtaining a sample (e.g., swabs), reagents for extracting DNA, enzymes (such as polymerase and/or ligase to capture a region of interest), reagents for amplifying the region of interest, reagents for purifying the DNA or amplified or captured regions of interest (e.g., purification cartridge), buffers, sequencing reagents, or any combination thereof. In one embodiment, the kit may be a low throughput kit, such as a kit for a small number of samples. For example, a kit may be a low throughput kit, such as a kit for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 20, 24, 28, 32, 36, 40, 42, 48, or between 8-48 samples. In another embodiment, the kit may be a high-throughput kit, such as a kit for a large number of samples. For example, a kit may be a high-throughput kit, such as a kit for 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, or more samples. For example, a kit may be a high-throughput kit, such as a kit for between 50-96, 50-384, 50-1536, 96-384, 96-1536, or 384-1536 samples. In some embodiments, a kit as described herein can comprise enough reagents to prepare one or more specimens for sequencing. For example, a kit as described herein can comprise enough reagents to prepare 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 48, 50, 60, 70, 80, 90, 96, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 384, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1536, 1750, 2000 or more specimens for sequencing.
Method of Using Probe Also provided herein is a method of using one or more probes disclosed herein, such as one or more probe set, for detecting, identifying, or distinguishing one or more organisms. The method can comprise identifying a an organism with a plurality of probes can detect at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, or 2000 different pathogens. In another embodiment, the plurality of probes can detect at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1250, 1500, 1750, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or more different strains, variants or sub-types of a pathogen or different strains or sub-types of different pathogens.
The method can comprise detecting or distinguishing different organisms, different pathogens, different strains, variants or sub-types of a pathogen or different strains, variants or sub-types of different pathogens, with at least 70% sensitivity, specificity, or both, such as with at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89% sensitivity, specificity, or both, such as with at least 90% sensitivity, specificity, or both. Each probe may detect or distinguish different organisms, different pathogens, different strains or sub-types of a pathogen or different strains or sub-types of different pathogens with at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sensitivity, specificity, or both, in an assay. Alternatively, a combination of probes may be used for detecting or distinguishing different organisms, different pathogens, different strains, variants or sub-types of a pathogen or different strains, variants or sub-types of different pathogens, with at least 70% sensitivity, specificity, or both, such as with at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89% sensitivity, specificity, or both, such as with at least 90% sensitivity, specificity, or both. Furthermore, the confidence level for determining the specificity, sensitivity, or both, may be with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% confidence.
In one embodiment, a method for detecting the presence of one or more target organisms is by contacting a sample suspected of containing at least one target organism with any of the probe set disclosed herein, capturing a region of interest of the at least one target organism (e.g., by polymerization and/or ligation) to form a circularized probe, and detecting the captured region of interest, thereby detecting the presence of the one or more target organisms.
In certain embodiments, the captured region of interest may be amplified to form a plurality of amplicons (e.g., by PCR). In some embodiments the sample is treated with nucleases to remove the linear nucleic acids after probe-circularizing capture of the region of interest. In some embodiments, the circularized probe is linearized, e.g., by nuclease treatment. In other embodiments the circularized probe molecule is sequenced directly by any means known in the art, without amplification. In certain embodiments, the circularized probe is contacted by an oligonucleotide that primes polymerase-mediated extension of the molecules to generate sequences complementary to that of the circularized probe, including from at least one to as many as 1 million or more concatemerized copies of the original circular probe.
In particular embodiments, the circularized probe molecule is enriched from the reaction solution by means of a secondary-capture oligonucleotide capture probe. A secondary-capture oligonucleotide capture probe may comprise a moiety designed to be captured, such as a biotin molecule, and a nucleic acid sequence designed to hybridize to at least 6 nucleotides of the circularized probe. The nucleic acid sequence designed to hybridize to at least 6 nucleotides of the circularized probe may include 1, 2, 4, 8, 16, 32 or more nucleotides of the polymerase-extended capture product.
In certain embodiments, the probe and/or captured region of interest is sequenced by any means known in the art, such as polymerase-dependent sequencing (including, dideoxy sequencing, pyrosequencing, and sequencing by synthesis) or ligase based sequencing (e.g., polony sequencing). The sequencing can be by Sanger sequencing or massive parallel sequencing, such as “next generation” (Next-gen) sequencing, second generation sequencing, or third generation sequencing. For example, sequencing can be by second generation or third generation sequencing methods, such as using commercial platforms such as Illumina, 454 (Roche), Solid, Ion Torrent PGM (Life Technologies), PacBio, Oxford, Life Technologies QDot, Nanopore, or any other available sequencing platform. Massive parallel sequencing can allow for the simultaneous sequencing of one million to several hundred millions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 48, 50, 60, 70, 80, 90, 96, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 million, of reads from amplified DNA clones. The reads can read any number of bases, such as 50-400 bases.
An internal nucleotide control, such as DNA at a known concentration, can be used with the methods and samples described herein. In one embodiment, an internal nucleotide control can serve as an internal calibrator, such as for determining copy number. In some embodiments, a sequencing read that aligns to the calibrator can also serve as a positive control for the performance of the assay, such as in the context of every sample.
In one aspect, the probes, methods, and kits described herein can be used to test for the presence of one or more organisms, such as those in Table 2. In one embodiment, the probes, methods, and kits described herein can be used to test for the presence of one or more antibiotic resistance genes, such as those in Table 3. In a preferred embodiment, the probes, methods, and kits described herein can be used to test for the presence of one or more organisms, such as those in Table 2, and test for the presence of one or more antibiotic resistance genes, such as those in Table 3, in parallel, such as in one sample tube, in the same sample, simultaneously, or any combination thereof. In some embodiments, in a single reaction tube, a kit can be used to test for the two or more microbes most commonly associated with hospital-acquired infections, and simultaneously tests for the presence of two or more antibiotic resistance genes. For example, a kit can be used to test for the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more microbes most commonly associated with hospital-acquired infections, and simultaneously tests for the presence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more antibiotic resistance genes simultaneously. For example, in a single reaction tube, a kit can be used to test for the 12 microbes most commonly associated with hospital-acquired infections, and simultaneously tests for the presence of 18 antibiotic resistance genes.
In one embodiment, one or more organisms can be identified from a sample, such as a sample form a host and the organism being identified is a pathogen. In one embodiment, the sample is a biological sample, such as from a mammal, such as a human. In another embodiment, a genotype of the host is identified or detected from the sample or another sample from the host. The identification of one or more organisms (such as one or more pathogens, such as different pathogens or subtypes or strains of pathogens), can be used to select one or more therapeutics or treatments for the host. In another embodiment, the identification of one or more organisms (such as one or more pathogens, such as different pathogens or subtypes or strains of pathogens), can be used to stratify the host into a therapeutic group, such as for a particular drug treatment or clinical trial. In one embodiment, HPV strain identification can be used to stratify a host into a cancer therapeutic group or to select a cancer treatment.
The yet another embodiment identification of one or more organisms (such as one or more pathogens, such as different pathogens or subtypes or strains of pathogens) and the genotype of a host can be used to select one or more therapeutics or treatments for the host. In another embodiment, the identification of one or more organisms (such as one or more pathogens, such as different pathogens or subtypes or strains of pathogens) and the genotype of the host can be used to stratify the host into a therapeutic group, such as for a particular drug treatment or clinical trial.
Also provided herein is a method for identifying an organism, such as a genetic signature of an organism, a subtype or strain of a pathogen in a short timeframe or with a fast turnaround time. In another embodiment, a genotype of an individual or host can also be identified within the short time frame. For example, the identification of a pathogen in a sample or the genotype of a host can completed in less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In one embodiment, from contacting the sample with one or more probes to identifying the organism by sequencing can be performed in less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In yet another embodiment, from contacting the sample with the probe to identifying the organism (such as one or more pathogens) by sequencing, and transmitting the results to a health care professional (such as a clinician or physician) can be performed in less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In yet another embodiment, from contacting the sample with the probe to identifying the organism (such as one or more pathogens) by sequencing, transmitting the results to a health care professional (such as a clinician or physician), and selection of a therapeutic can be performed in less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.
Also provided herein is a method for simultaneous quantification and identification of an organism, such as identifying one or more subtypes or substrains of a pathogen. Multiplexing is also provided herein, wherein a multiple pathogens, substrains or subtypes of pathogens, can be detected simultaneously or in a single reaction tube.
In one embodiment, conversion of sequence data to quantitative report can be performed by using selected validated parameters. Any software known in the arts can be used for any of the methods disclosed herein.
In some embodiments, an organism identified and/or quantified using the methods described herein can be the cause of an infection in a subject, such as a nosocomial infection (also known as a hospital-acquired infection (HAI)) which is an infection whose development is favored by a hospital environment. In some embodiments, an infection can be acquired by a patient during a hospital visit or one developing among hospital staff. Such infections can include, for example, fungal and bacterial infections and can be aggravated by a reduced resistance of individual patients. Organisms responsible for HAIs can survive for a long time on surfaces in the hospital and can enter or be transmitted to the body through wounds, catheters, and ventilators. In some embodiments, the route of transmission can be contact transmission (direct or indirect), droplet transmission, airborne transmission, common vehicle transmission, vector borne transmission, or any combination thereof.
People in hospitals can already be in a poor state of health, impairing their defense against bacteria. Advanced age or premature birth along with immunodeficiency, due to, for example, drugs, illness, or irradiation, present a general risk. Other diseases can present specific risks, for example, chronic obstructive pulmonary disease can increase chances of respiratory tract infection. Invasive devices, for example, intubation tubes, catheters, surgical drains, and tracheostomy tubes can bypass the body's natural lines of defense against pathogens and can provide an easy route for infection. Patients already colonized on admission can be put at greater risk when they undergo a procedure, such as an invasive procedure. A patient's treatment itself can leave the patient vulnerable to infection, for example, immunosuppression and antacid treatment can undermine the body's defenses, while antimicrobial and recurrent blood transfusions can also be risk factors.
Non-limiting examples of HAIs include Ventilator associated pneumonia (VAP), Staphylococcus aureus, Methicillin resistant Staphylococcus aureus (MRSA), Candida albicans, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Clostridium difficile, Tuberculosis, Urinary tract infection, Hospital-acquired pneumonia (HAP), Gastroenteritis, Vancomycin-resistant Enterococcus (VRE), and Legionnaires' disease. In some embodiments, HAIs can be caused by one or more of the organisms provided in Table 2.
Nucleotides, such as DNA and RNA, can be isolated from any suitable sample and detected using the probes described herein. Non-limiting examples of sample sources include catheters, medical devices, blood, blood cultures, urine, stool, fomites, wounds, sputum, pure bacterial cultures, mixed bacterial cultures, and bacterial colonies.
In some embodiments, the probe sets described herein can be used to detect and distinguish among the organisms responsible for more than 10% of the hospital acquired infections at a site. For example, the probe sets described herein can be used to detect and distinguish among the organisms responsible for more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the hospital acquired infections at a site. In some embodiments, a site can be a surgical site, wound, tract, urinary catheter, ventilator, intravenous needle, syringe, respiratory tract, invasive device, intubation tube, catheter, surgical drain, tracheostomy tube, saline flush syringe, vial, bag, tube or any combination thereof.
Method of Generating Probe A further aspect of the invention provides methods of making the mixtures of probes provided by the invention. The methods comprise providing a set of reference genomes and an exclusion set of genomes. The sequence of the reference genomes can be partitioned (in silico) into n-mer strings of about 18-50 nucleotides. The partitioned n-mer strings can be screened to eliminate redundant sequences, sequences with secondary structure, repetitive sequences (e.g., strings with more than 4 consecutive identical nucleotides), and sequences with a Tm outside of a predetermined range (e.g., outside of 50-72° C.). The screened n-mers can be further screened to identify homologous probe sequences by eliminating n-mers that specifically hybridize to a sequence in the genome in the exclusion set of genomes (e.g., if a pairwise alignment contains 19 of 20 matches in an n-mer, such as a 25-mer) or occurs in the genome of the target organism more than a specified number of times. The screening may also remove n-mers that are present in more than or less than a specified number of the reference genomes. The screening may also remove n-mers that will not interact favorably with enzymes to be used with the probe sequences. For example, a particular polymerase may work with higher efficiency if the last 3′ base of the probe is a G or C. Similarly, a particular ligase may work more efficiently on certain bases at the ligation junction. For example, Ampligase (Epicentre) will ligate a gap between AG and GT at least 10 times more efficiently than a gap between TC and CC.
In particular embodiments, a homologous probe sequence may occur only once in the genome of the target organism. For target organisms with a single-stranded genome, the homologous probe sequence may occur only once in the complement of the genome of the target organism. In one embodiment, where a sequenced variant of the target organism is available (e.g., the same species, genus, or serovar), the homologous probe sequences can be filtered so as to specifically hybridize to the genome of the additional sequenced variant(s) resulting in a probe that groups related organisms. In an alternate embodiment, the homologous probe sequences can be filtered so as to not specifically hybridize to the genome of the sequenced variant (e.g., the sequenced variant is part of the exclusion set), resulting in a probe that discriminates between related organisms. These filter processes can be iterated for each target organism to be detected by the particular mixture. In some embodiments, the candidate homologous probe sequences can be screened to eliminate those that will specifically hybridize with other probes in the mixture.
Probe selection can be based on a database of different pathogens, strains of a pathogen, or both, such as a database comprising more than 10 different pathogens, strains of a pathogen, or both. For example, probe selection can be based a database comprising more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, or more different pathogens, strains of a pathogen, or both. In some embodiments, probe selection can be based on a database of different pathogens, strains of a pathogen, or both, that are known to cause HAIs, such as a database comprising more than 10 different pathogens, strains of a pathogen, or both, that are known to cause HAIs. For example, probe selection can be based a database comprising more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, or more different pathogens, strains of a pathogen, or both, that are known to cause HAIs, and optionally with additional strains or sub-types of other pathogens. In one embodiment, probes for organisms associated with HAIs are selected by partitioning all available genomes of organisms associated with HAIs into one or more subsets based on sequence similarity. For each subset candidate probe sets are generated that capture all strains. A filter can then be applied for specificity against human/microbial/viral/fungal genomes.
Some of clinical tests based on the methods disclosed herein rely on the ability to determine or approximate the number of input template molecules (genomes) in a sample. A two step method can be used to calculate the number of template molecules in a sample from the sequencing read counts. 1) Each sample sequenced can have a known quantity of a control sequence added to it. One embodiment employs GFP as the control sequence. It is contemplated to use several control sequences added in different quantities. The first step in analyzing sequencing reads can be to normalize the counts based on the number of reads that came from the control sequence. This normalization accounts for the fact that more material from sample A than from sample B may have been put into the sequencing reaction. 2) Since different MIPs (or primer pairs or hybridization capture probes) might work with different efficiencies, the second step of the quantification process can be to normalize between probes. In one embodiment, this normalization relies on experiments in which fixed amounts of different templates were sequenced and might reveal, e.g., that a probe against one strain or organism produces 2 circularized MIPs per template but a probe against anther strain or organism produces 3. Thus, the count for the first probe might be multiplied by 33.3 and the count for the second probe divided by 50 to produce comparable load counts for the two strains.
Some embodiments use a mixed quantity of GFP as the control sequence and a variable quantity of one or more organisms or strains. Some samples may contain only GFP and template DNA while others also included a human background. After the sequencing reads are separated by sample, the method can calculate the ratio of reads, such as viral (HPV-18, HIV-CN006, and HIV-CN009) reads, to GFP and plots that ratio against the number of template molecules in the reaction. Those plots indicate generally excellent agreement between the viral/GFP ratio and the input template quantity.
Compared to other assays, high throughput sequencing offers a relatively unique ability to detect and genotype the pathogen DNA and the human DNA in a sample from a single reaction. In current clinical practice, genotyping the pathogen and human may require multiple tests, potentially doubling (or more) the expense compared to simply detecting a pathogen. The methods disclosed herein enable simultaneous genotyping with minimal added cost and often no added labor. Other selection/enrichment technologies would also enable these tests.
The methods disclosed herein provide for simultaneously detecting or genotyping multiple pathogens.
For example, the methods provide for: coinfection of HIV and HCV, simultaneously genotyping/quantifying HIV while testing for diseases common in immunocompromised patients. Doctors typically only test for diseases like Candida, CMV, etc upon presentation of some other symptom. However, if the tests can be added at minimal cost, this might be a unique market and feature for Pathogenica's product, for example, HPV and other STIs. There is an interest in testing for HPV and other STIs, primarily chlamydia and gonorrhea to simplify screening, especially in patient populations with limited access to doctors. There is also an interest in testing for these diseases as additional risk factors for cervical cancer.
Probe Panel Table 1 lists the probe arm sequences in one embodiment of the present invention designed to detect a variety pathogenic organisms, such as those provided in Table 2, from a sample. Non limiting examples include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Acinetobacter baumanii, Clostridium difficile, Escherichia coli, Enterobacter (aerogenes, cloacae, asburiae), Enterococcus (faecium, faecalis), Klebsiella pneumoniae, Proteus mirabilis, Candida albicans, and Pseudomonas aeruginosa. The probe set can also be used to detect many common drug resistance genes, including, but not limited to CARB, CMY, CTX-M, GES, IMP, KPC, NDM, Other ampC, OXA, PER, SHV, VEB, VIM, ermA, vanA, vanB, mecA, and mexA,
Tables 1 and 3-14 provide regions of interest (leftmost columns, using the format of descriptor (e.g., organism or gene, if applicable)_reference accession number (if applicable)_first nucleotide of capture region_last nucleotide of capture region. For example, the probe “acinetobacter_NC—010611—627997—628164” is directed to acineobacter, and is predicted to be capable of capturing nucleotides corresponding to nucleotides 627997 to 628164 of the reference sequence NC—010611. Reference accession sequences can be obtained from, for example, the NCBI Entrez portal. Tables 3, 5, 7, 9, 11, and 13 provide the regions of interest and corresponding annotated genes within that region. Tables 4, 6, 8, 10, 12, and 14, in turn, provide particular exemplary oligonucleic acid sequences—provided as pairs that can be used in a MIP or adapted for use as conventional PCR primers—predicted to capture the region of interest listed in the first column of the. “Binding region 1” in Tables 4, 6, 8, 10, 12, and 14 correspond to the 5′, or ligation arm, of a MIP probe and “Binding region 2” corresponds to the 3′, or extension arm of a MIP probe. In some embodiments, substantially similar sequences to the regions of interest provided in Tables 1 and 3-14 can be used. In some embodiments, the substantially similar sequences wherein the substantially similar sequences are 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 100% identical to the sequence of the regions of interest. In other embodiments, the substantially similar sequences have endpoints within 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides upstream or downstream of either of the endpoints of the regions of interest. In still other embodiments, the substantially similar sequences are 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 100% identical to the sequence of the regions of interest and have endpoints within 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides upstream or downstream of either of the endpoints of the regions of interest. In still more particular embodiments, the particular exemplified endpoints and binding regions are use, e.g., as pairs of binding regions in either a single MIP capture probe, or as pairs of conventional PCR primers, e.g., using the reverse complement of the ligation arm.
Subsets of the regions of interest or particular exemplary binding regions in tables Tables 1 and 3-14 can be used concordant with the present invention, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of the regions of interest or binding regions in the tables, e.g.:
oligonucleic acid molecules capable of i) amplifying, geometrically by polymerase chain reaction or ii) circularizing capture of 1, 2, 3, 4, 5, 10, 15, 16, or all 17, of the regions of interest provided in column 1 of Table 3, or substantially similar sequences;
oligonucleic acid molecules capable of i) amplifying, geometrically by polymerase chain reaction or ii) circularizing capture of 1, 2, 3, 4, 5, 10, 15, 20, 30, 50, 100, or all 134, of the regions of interest provided in column 1 of Table 5, or substantially similar sequences, such as:
oligonucleic acid molecules capable of i) amplifying, geometrically by polymerase chain reaction or ii) circularizing capture of, 1, 2, 3, 4, 5, 10, or all 13, of the regions of interest provided in column 1 of Table 7, or substantially similar sequences;
oligonucleic acid molecules capable amplifying, geometrically by polymerase chain reaction, or circularizing capture of, 1, 2, 3, 4, 5, 10, 20, 40, 60, 80, or all 85, of the regions of interest provided in column 1 of Table 9, or substantially similar sequences;
oligonucleic acid molecules capable of i) amplifying, geometrically by polymerase chain reaction or ii) circularizing capture of, 1, 2, 3, 4, 5, 10, 20, 25, or all 29 of the regions of interest provided in column 1 of Table 11, or substantially similar sequences;
oligonucleic acid molecules capable of i) amplifying, geometrically by polymerase chain reaction or ii) circularizing capture of, 1, 2, 3, 4, 5, 10, 15, or all 20, of the regions of interest provided in column 1 of Table 13, or substantially similar sequences;
oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, or all 34 of the sequences, or reverse complements thereof, provided in the second or third column of table 4;
oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 20, 50, 100, 150, 200, 250, or all 268 of the sequences, or reverse complements thereof, provided in the second or third column of table 6;
oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 15, 20, 25, or all 26 of the sequences, or reverse complements thereof, provided in the second or third column of table 8;
oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 20, 50, 100, 150, or all 170 of the sequences, or reverse complements thereof, provided in the second or third column of table 10;
oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 20, 30, 40, 50, or all 56 of the sequences, or reverse complements thereof, provided in the second or third column of table 12;
oligonucleic acid molecules comprising 1, 2, 4, 6, 8, 10, 20, 30, or all 40 of the sequences, or reverse complements thereof, provided in the second or third column of table 14, as well as any combinations of the foregoing.
Table 1 provides particular probes assembled as molecular inversion probes (MIPs) capable of circularizing capture of the indicated region of interest in the leftmost column. These exemplary probes share a common backbone sequence of GTTGGAGGCTCATCGTTCCTATATTCCACACCACTTATTATTACAGATGTTATGCT CGCAGGTC, except for the peGFP_N1—730—925 probe, which uses the backbone GTTGGAGGCTCATCGTTCCTATATTCCTGACTCCTCATTGATGATTACAGATGTTA TGCTCGCAGGTC. Alternative backbone sequences can readily be used. Conventional PCR primer pairs can be adapted from these MIP probes by omitting the intervening backbone sequence and providing the reverse complement of the ligation arm (5′) probe. Tables 4, 6, 8, 10, 12, and 14 provide subsets of the probes in Table 1 where the individual arms are provided in the second and third columns, respectively. Tables 4, 6, 8, 10, 12, and 14 collectively provide the same probe arms that are present in Table 1.
TABLE 1
A particular embodiment of the probe sets provided by the invention
Name Sequence
peGFP_N1_730_925 /5Phos/GTGGTATGGCTGATTATGATCTAGAGTGTTGGAGGCTCATCGTTCCTATA
TTCCTGACTCCTCATTGATGATTACAGATGTTATGCTCGCAGGTCGAGTTTGGACAA
ACCACAACTAGAA
plasmids_NC_010660_187035_187205 /5Phos/GCTGTCACCGTCCAGACGCTGTTGGCGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCCGTGCCTTCAAGCGCG
plasmids_NC_014232_5501_5677 /5Phos/GACTCCGCAGAATACGGCACCGTGCGCAGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCGTACAGGCCAGTC
AGC
plasmids_NC_011980_58308_58487 /5Phos/GCAGTCGGTAACCTCGCGCGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCGCGCTATCTCTGCTCTCACTGC
plasmids_NC_011838_178818_178996 /5Phos/GCTGTCCTGGCTGCAAGCCTGGGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCGAACTGCTGATGGACGT
plasmids_FN554767_13017_13190 /5Phos/GACAGCAGACTCACCGGCTGGTTCCGCTGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCAAGATGCTGCTGG
CCACACTG
plasmids_NC_013655_115365_115542 /5Phos/GACAGAACAAGTTCCGCTCCGGGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACGGATACGCCGCGCAT
plasmids_NC_013950_90185_90338 /5Phos/GAGGACCGAAGGAGCTAACCGGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGCCGCATACACTATTCTC
plasmids_NC_015599_37281_37455 /5Phos/GCTGTAATGCAAGTAGCGTATGCGCTCGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAACAGCAAGGCCGCC
AATGCCTGACG
plasmids_NC_013951_69899_70067 /5Phos/GAACGTCTGGCGCTGGTCGCCTGCCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCACAGGTGCTGACGTGGT
plasmids_NC_007351_37979_38146 /5Phos/CGCATATGCTGAATGATTATCTCGTTGCGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTTGCTCAATGAG
GTTATTCA
plasmids_FN822749_1846_2009 /5Phos/GACGACAGATGCAGGTTGAGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCCGCATCGCCGATGCTCATC
plasmids_NC_004851_143949_144109 /5Phos/CGCCTGCTCCAGTGCATCCAGCACGAATGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATGCTCTCCGCCATC
GCGTTGTCA
plasmids_NC_010558_156799_156957 /5Phos/AGTGCGTTCACCGAATACGTGCGCAGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCAGGTTATGCCGCTCAAT
TC
plasmids_NC_007635_38395_38566 /5Phos/AATCCAGGTCCTGACCGTTCTGTCCGTGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACCTCCGTTGAGCTGA
TGGA
plasmids_NC_009787_17946_18116 /5Phos/GAGGTGGCCAACACCATGTGTGACCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGACGCCGGTATATCGGTA
TCGAGCTGCT
plasmids_NC_012547_53585_53752 /5Phos/CGCATATGCTGAATGATTATCTCGTTGGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGGTGATCTTGCTCA
ATGAGGTTATTC
plasmids_NC_006671_56259_56438 /5Phos/GAAGTGCCGGACTTCTGCAGAGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCACGGCCTGATGGAGGCCGC
plasmids_NC_014385_53151_53310 /5Phos/GCTAATCGCATAACAGCTACGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCCATCACGTAACTTATTGATGATA
TT
plasmids_FN649418_57169_57339 /5Phos/GCTGCGGTATTCCACGGTCGGCCGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCAGGAACGCTGCCTGTGGTC
plasmids_NC_005011_8620_8785 /5Phos/GAATCAATTATCTTCTTCATTATTGATGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTGCGGCTCAACTCAA
GCA
plasmids_NC_014843_98413_98578 /5Phos/GTCACACGTCACGCAGTCCGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCGCATTCATGGCGCTGATGGC
plasmids_NC_008490_5165_5334 /5Phos/GTGTTACTCGGTAGAATGCTCGCAAGGGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTAGATGACATATCA
TGTAAGTT
plasmids_NC_015963_147516_147686 /5Phos/CGGAACTGCCTGCTCGTATGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCAACGATATAGTCCGTTAT
plasmids_NC_007365_100545_100708 /5Phos/GCTCTCCGACTCCTGGTACGTCAGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCGCGCATTAATGAAGCAC
plasmids_NC_009838_104163_104332 /5Phos/GATGTTGCGATTACTTCGCCAACTATTGGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTGTAATTATGACG
ACGCCG
plasmids_NC_013452_4052_4209 /5Phos/CTCATTCCAGAAGCAACTTCTTCTTGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGATAGCCATGGCTACAA
GAATA
plasmids_NC_010409_39768_39935 /5Phos/GCAATACCAGGAAGGAAGTCTTACTGGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTCATTGGAGAACAGAT
GATTGATGT
plasmids_NC_014233_50337_50492 /5Phos/GTATCGCCACAATAACTGCCGGAAGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAACGATATAGTCCGTTATG
plasmids_NC_013950_91008_91174 /5Phos/GCTGTGGCACAGGCTGAACGCCGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGTGATGTCATTCTGGTTAA
GA
plasmids_NC_002698_168967_169123 /5Phos/ACATAATCTGAATCTGAGACAACATCGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGCACTCTGGCCACAC
TGG
plasmids_NC_013362_56651_56805 /5Phos/GTGAAGCGCATCCGGTCACCGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCATGGCATAGGCCAGGTCAATAT
plasmids_NC_014208_52313_52469 /5Phos/GGTTCTGGACCAGTTGCGTGAGCGCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTAACATCGTTGCTGCT
CCAT
betalactamase_AB372224_738_905 /5Phos/CGCTGGATTTCACGCCATAGGCGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTCGCTACCGTTGATGATT
betalactamase_EF685371_398_548 /5Phos/CGTATAGGTGGCTAAGTGCAGCGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTAACTCATTCCTGAGGGTTTC
betalactamase_DQ149247_231_371 /5Phos/GTACATACTCGATCGAAGCACGAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCGGAATAGCGGAAGCTTTC
betalactamase_AY750911_244_414 /5Phos/AAGGTCGAAGCAGGTACATACTCGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGACATGAGCTCAAGTCCA
AT
betalactamase_DQ519087_417_575 /5Phos/GAAGCTTTCATAGCGTCGCCTAGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTAGCTAGCTTGTAAGCAAA
TTG
betalactamase_AM231719_379_537 /5Phos/GAAGCTTTCATGGCATCGCCTAGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCTAGCTTGTAAGCAAACTG
betalactamase_Y14156_663_819 /5Phos/CGCTACCGGTAGTATTGCCCTTGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGAATATCCCGACGGCTTTC
betalactamase_JN227085_763_931 /5Phos/ATCGCCACGTTATCGCTGTACTGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTTACCCAGCGTCAGATTCC
betalactamase_EU259884_1030_1170 /5Phos/CAAGTACTGTTCCTGTACGTCAGCGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCGCCAGTAACTGGTCTAT
TC
betalactamase_HQ913565_578_730 /5Phos/CAACGTCTGCGCCATCGCCGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCCGCAATATCATTGGTGGTGC
betalactamase_AY524988_385_552 /5Phos/GCCGCCCGAAGGACATCAACGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCCAGACGGGACGTACACAAC
CARB_AF030945_646_795 /5Phos/CGTGCTGGCTATTGCCTTAGGGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTAATACTCCTAGCACCAAATC
CARB_U14749_1227_1390 /5Phos/CATTAGGAGTTGTCGTATCCCTCAGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATACTCCGAGCACCAAATC
CARB_AF313471_2731_2906 /5Phos/AAATTGCAGTTCGCGCTTAGCGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTCCATAGCGTTAAGGTTTC
CMY_DQ463751_613_790 /5Phos/GCGCCAAACAGACCAATGCTGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCGATTTCACGCCATAGGCTC
CMY_EF685371_397_552 /5Phos/GTATAGGTGGCTAAGTGCAGCAGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCGTAACTCATTCCTGAGGG
CMY_EU515251_583_733 /5Phos/GTCATCGCCTCTTCGTAGCTCGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCATATCGATAACGCTGG
CMY_X92508_126_301 /5Phos/AGTATCTTACCTGAAATTCCCTCACGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCTCTCGTCATAAGTCGA
ATG
CMY_AB061794_343_489 /5Phos/CATCACGAAGCCCGCCACAGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCGCCCTTGAGCGGAAGTATC
CMY_JN714478_1882_2055 /5Phos/ACCAATACGCCAGTAGCGAGAGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCAACGTAGCTGCCAAATC
CMY_X91840_1872_2046 /5Phos/CAATCAGTGTGTTTGATTTGCACCGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTACCCGGAATAGCCTGCTC
CTXM_EF219134_13713_13858 /5Phos/CGGATAACGCCACGGGATGAGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCACCGGGTCAAAGAATTCCTC
CTXM_HQ398215_802_947 /5Phos/GCGGCGTGGTGGTGTCTCGTTGGAGGCTCATCGTTCCTATATTCCACACC
ACTTATTATTACAGATGTTATGCTCGCAGGTCCGCTGCCGGTCTTATCAC
CTXM_AM982522_639_788 /5Phos/GCCACGTCACCAGCTGCGGTTGGAGGCTCATCGTTCCTATATTCCACACC
ACTTATTATTACAGATGTTATGCTCGCAGGTCCGGCTGGGTGAAGTAAGTC
GES_HM173356_1163_1321 /5Phos/GCTCGTAGCGTCGCGTCTCGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCTTGACCGACAGAGGCAAC
GES_AF156486_1754_1905 /5Phos/CAGCAGGTCCGCCAATTTCTCGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGTGGACGTCAGTGCGC
GES_HQ874631_571_748 /5Phos/CCATAGAGGACTTTAGCCACAGTGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTACACCGCTACAGCGTAAT
GES_FJ820124_1174_1338 /5Phos/CATATGCAGAGTGAGCGGTCCGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCAATTCTTTCAAAGACCAGC
IMG_DQ361087_489_645 /5Phos/CCATTAACTTCTTCAAACGATGTATGGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACCCGTGCTGTCGCTAT
IMG_JN848782_301_475 /5Phos/GTGCTGTCGCTATGGAAATGTGGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCAACCAAACCACTAGGTTATCTT
IMG_EF192154_182_328 /5Phos/GTCAGTGTTTACAAGAACCACCAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATGCATACGTGGGAATAGATT
IMG_AY033653_1343_1500 /5Phos/CGGAAGTATCCGCGCGCCGTTGGAGGCTCATCGTTCCTATATTCCACACC
ACTTATTATTACAGATGTTATGCTCGCAGGTCTTCGATCACGGCACGATC
IMG_AF318077_871_1047 /5Phos/CGAACCAGCTTGGTTCCCAAGGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCACTGCGTGTTCGCTC
IMG_AF318077_515_657 /5Phos/GATGCTGTACTTTGTGATGCCTAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGCTTGGCAAGTACTGTTC
KPC_HM066995_226_375 /5Phos/GCAAGAAAGCCCTTGAATGAGCGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCGTTATCACTGTATTGCAC
KPC_GQ140348_624_799 /5Phos/AATCAACAAACTGCTGCCGCTGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTGTACTTGTCATCCTTGT
KPC_EU729727_683_840 /5Phos/CCAGTCTGCCGGCACCGCGTTGGAGGCTCATCGTTCCTATATTCCACACC
ACTTATTATTACAGATGTTATGCTCGCAGGTCTCGAGCGCGAGTCTAGC
KPC_FJ234412_691_839 /5Phos/CCGACTGCCCAGTCTGCCGGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCCGAGCGCGAGTCTAGCC
NDM_JN104597_64_211 /5Phos/GTAAATAGATGATCTTAATTTGGTTCACGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTGCTGGCCAATCGT
CG
NDM_FN396876_2744_2885 /5Phos/CACAGCCTGACTTTCGCCGCGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCCAAGCAGGAGATCAACCTGC
NDM_FN396876_2958_3117 /5Phos/GGTGGTCGATACCGCCTGGGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCGTGAAATCCGCCCGACG
NDM_JN104597_314_465 /5Phos/CATGTCGAGATAGGAAGTGTGCGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGATGCGCGTGAGTCAC
NDM_FN396876_2382_2548 /5Phos/CAATCTGCCATCGCGCGATTGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCCGGCAATCTCGGTGATGC
OXA_EF650035_239_388 /5Phos/CGAAGCAGGTACATACTCGGTCGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGAGCTAAATCTTGATAAAC
TT
OXA_EU019535_389_537 /5Phos/TAGAATAGCGGAAGCTTTCATGGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCTAGCTTGTAAGCAAACTG
OXA_EF650035_423_594 /5Phos/CAAGTCCAATACGACGAGCTAAAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAATAGCATGGATTGCACTTC
OXA_DQ309276_232_380 /5Phos/GGTACATACTCGGTCGAAGCACGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATCTTGATAAACTGAAATAG
CG
OXA_DQ445683_232_380 /5Phos/GGTACATACTCGGTCGATGCACGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCTTGATAAACCGGAATAGCG
OXA_X75562_201_366 /5Phos/GTAATTGAACTAGCTAATGCCGTACGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTATGACACCAGTTTCTA
GGC
OXA_M55547_995_1154 /5Phos/CAAGTACTGTTCCTGTACGTCAGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCCAGTTGTGATGCATTC
OXA_AY445080_313_469 /5Phos/TCTCTTTCCCATTGTTTCATGGCGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGCGGAAATTCTAAGCTGAC
PER_Z21957_217_371 /5Phos/GTAGGTTATGCAGTTATTAGGTTCAGGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGACTCAGCCGAGTCAAGC
PER_HQ713678_6002_6167 /5Phos/GCAGTACCAACATAGCTAAATGCGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAATAACAAATCACAGGCCAC
PER_GQ396303_667_844 /5Phos/GGTCCTGTGGTGGTTTCCACCGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGCGATAATGGCTTCATTGG
PER_X93314_954_1122 /5Phos/TAACCGCTGTGGTCCTGTGGGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCTGCGCAATAATAGCTTCATTG
PER_HQ713678_4517_4674 /5Phos/GGAAGCGTTGCTTGCCATAGTGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAACCGAAGCACCATGTAATT
PER_HQ713678_5074_5219 /5Phos/GTTCGGTGCAAAGACGCCGGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCTCGCAGACTTCAATATCAATATT
PER_GQ396303_254_399 /5Phos/CACCTGATGCAGAACCAGCATGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGGCCACGTTATCACTGTG
SHV_AY661885_656_806 /5Phos/CAGCTGCCGTTGCGAACGGTTGGAGGCTCATCGTTCCTATATTCCACACC
ACTTATTATTACAGATGTTATGCTCGCAGGTCCGCAGATAAATCACCACAATC
SHV_AF535128_587_761 /5Phos/GCTCAGACGCTGGCTGGTCGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCCCGCAGATAAATCACCACG
SHV_U92041_406_579 /5Phos/GCCAGTAGCAGATTGGCGGCGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCGAACGGGCGCTCAGACG
SHV_AY288915_617_764 /5Phos/CCACTGCAGCAGATGCCGTGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCGTATCCCGCAGATAAATCACC
SHV_HQ637576_88_245 /5Phos/TTAATTTGCTTAAGCGGCTGCGGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCAGCTGTTCGTCACCG
SHV_AF535128_188_362 /5Phos/GGGAAAGCGTTCATCGGCGGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCTCGCTCATGGTAATGGCG
SHV_X98102_763_913 /5Phos/TCTTATCGGCGATAAACCAGCCGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTTGCCAGTGCTCGAT
TEM_X64523_2037_2191 /5Phos/CAGTCCCTCGATATTCAGATCAGAGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTAACAATTTCGCAACCGTC
TEM_J01749_2068_2239 /5Phos/CAGCTGCGGTAAAGCTCATCAGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATAGTTAAGCCAGTATACACTC
TEM_GQ149347_3605_3747 /5Phos/GTCGGAAAGTTGACCAGACATTAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATACTAGGAGAAGTTAATAA
ATACG
TEM_U36911_4374_4551 /5Phos/CATTCTCTCGCTTTAATTTATTAACCTGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCGACCTTCTGGACA
TTATC
TEM_AF091113_1529_1699 /5Phos/GTAACAACTTTCATGCTCTCCTAAAGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGGTAACTGATGCCGTAT
TT
TEM_GU371926_11801_11944 /5Phos/GTGAAGTGAATGGTCAGTATGTTGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGTGCGCAGGAGATTAGC
TEM_J01749_766_908 /5Phos/CCTGTCCTACGAGTTGCATGATGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCATAATGGCCTGCTTCTCGC
TEM_J01749_1634_1783 /5Phos/CGTTTCCAGACTTTACGAAACACGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGTTGTGAGGGTAAACAAC
TEM_U36911_7596_7762 /5Phos/CGTTGCTTACGCAACCAAATATCGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGATCTTGCTCAATGAGGTTA
TEM_U36911_6901_7069 /5Phos/CATCATGTTCATATTTATCAGAGCTCGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTAGATTTCATAAAGTCT
AACACAC
TEM_GU371926_33909_34082 /5Phos/GTTTCCACATGGTGAACGGTGGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAACCTGTCACTCTGAATGTT
VEB_EU259884_6947_7094 /5Phos/CAAATACTAAATTATACAGTATCAGAGAGGTTGGAGGCTCATCGTTCCTA
TATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATGCAAAGCGTTAT
GAAATTTC
VEB_EF136375_596_738 /5Phos/GTTCTTATTATTATAAGTATCTATTAACAGTTGTTGGAGGCTCATCGTTC
CTATATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATTAGTGGCT
GCTGCAAT
VEB_EF420108_234_380 /5Phos/CATCGGGAAATGGAAGTCGTTATGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTCAATCGTCAAAGTTGTTC
VEB_AF010416_89_230 /5Phos/CGTGGTTTGTGCTGAGCAAAGGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCAAAGTTAAGTTGTCAGTTTGAG
VIM_AY524988_385_552 /5Phos/GCCGCCCGAAGGACATCAAGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCAGACGGGACGTACACAAC
VIM_Y18050_3464_3614 /5Phos/GCAACTCATCACCATCACGGAGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGATGCGTACGTTGCCAC
VIM_AY635904_58_203 /5Phos/GCGACAGCCATGACAGACGCGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCGGACAATGAGACCATTGGAC
VIM_HM750249_275_454 /5Phos/AAACGACTGCGTTGCGATATGGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTCCGAAGGACATCAACGC
VIM_AJ536835_313_481 /5Phos/ATGCGACCAAACGCCATCGCGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCATCGTCATGGAAGTGCGTA
VIM_EU118148_131_300 /5Phos/GAACAGGCTTATGTCAACTGGGGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATAACATCAAACATCGACCC
VIM_DQ143913_921_1063 /5Phos/ACGAACCGAACAGGCTTATGTCGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTAACGCGCTTGCTGCTT
VIM_EU118148_2821_2961 /5Phos/GCTGTAATTATGACGACGCCGGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTCGGTGAGATTCAGAATGC
VIM_EU118148_1060_1229 /5Phos/CATCATAGACGCGGTCAAATAGAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTCATCACCATCACGGAC
van_DQ018710.1_6481_6652 /5Phos/GTGTATGTCAGCGATTTGTCCATGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTCATATTGTCTTGCCGATT
van_DQ018710.1_6764_6926 /5Phos/GTCCACCTCGCCAACAATCAAGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCATATCAACACGGGAAAGACCT
van_AY926880.1_3640_3785 /5Phos/GCGTGATTATCACGTTCGGCAGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTGCAGATTTAACCGACAC
van_FJ545640.1_517_690 /5Phos/GGCTCGACTTCCTGATGAATACGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGAAACCGGGCAGAGTATT
van_AE017171.1_34715_34859 /5Phos/CAACGATGTATGTCAACGATTTGTGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATTGCGTAGTCCAATTCGTC
van_NC_008821.1_11898_12045 /5Phos/CAGGCTGTTTCGGGCTGTGAGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCGGGTTATTAATAAAGATGATAGGC
van_FJ349556.1_5601_5765 /5Phos/GGCTCGGCTTCCTGATGAATACGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGGCATGGTATTGACTTCATT
mecA_AY820253.1_1431_1608 /5Phos/TAATTCAAGTGCAACTCTCGCAAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTTATTCTCTAATGCGCTAT
ATATT
mecA_AY952298.1_130_302 /5Phos/GGATAGTTACGACTTTCTGCTTCAGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTATTGCTATTATCGTCA
ACG
mecA_AM048806.2_1574_1720 /5Phos/CAGTATTTCACCTTGTCCGTAACCGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTTACGACTTGTTGCATGC
mecA_EF692630.1_239_405 /5Phos/AATGTTTATATCTTTAACGCCTAAACTGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATGCTTTGGTCTTTCT
GCAT
mex_AF092566.1_371_520 /5Phos/CTGGCCCTTGAGGTCGCGGGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCCGGTCTTCACCTCGACAC
mex_AF092566.1_50_193 /5Phos/GACGTAGATCGGGTCGAGCTGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCACGGAAACCTCGGAGAATT
mex_CP000438.1_487178_487357 /5Phos/GGCGTACTGCTGCTTGCTCAGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCTGACGTCGACGTAGATCG
mex_NZ_AAQW01000001.1_461304_461466 /5Phos/CCTGTTCCTGGGTCGAAGCCGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTCGGTCACCGCGGA
erm_NC_002745.2_871803_871973 /5Phos/GTCAGGCTAAATATAGCTATCTTATCGGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCAGTTACTGCTATAG
AAATTGAT
erm_NC_002745.2_871666_871841 /5Phos/CATCCTAAGCCAAGTGTAGACTCGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAGATATATGGTAATATTCC
TTATAAC
erm_EU047809.1_79_229 /5Phos/GTTTATAAGTGGGTAAACCGTGAATGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAAACGAGCTTTAGGTTT
GC
acinetobacter_NC_010611_627997_628164 /5Phos/GCAGCACTTGACCGCCATGAGTGACCAGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATCGCACCAACAACA
ATAATCG
acinetobacter_NC_010611_2417580_2417755 /5Phos/GTGATCACTGATGCACCAGATGAAGTGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTTGATATTCAAGTC
TATGACG
acinetobacter_CP002522_11753_11931 /5Phos/GATATTATTGATCATGGTGCCAAGCCAAGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCAATATGAAGCTGAC
GACGCG
acinetobacter_NC_011586_3908329_3908508 /5Phos/GCTGAGCGTGAAGGTTCATGGATTATTAGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGTAAGGCTTACGGT
CTCAT
acinetobacter_NC_010611_145181_145340 /5Phos/GCATCTTGTGCAGCCTGAATAGCAGCGTGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACCACGTTGAATATC
ACCTTCGGCAT
acinetobacter_NC_010611_3854494_3854662 /5Phos/AAGTCCATAATTGCTTGAGTGTAGTCATGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTTCGCACTGAAT
AATAAGAACAT
acinetobacter_NC_010400_56216_56383 /5Phos/GCTTGCTGGTTCTGCACGTAGCTTACTGGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAGATGAACAGGCTA
CTGCAA
acinetobacter_NC_010611_1454960_1455136 /5Phos/GCAGCGCTGTGCAAGTTCAATGTATTCTGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTCGTGCGAGTATTC
CTTAAGTGT
acinetobacter_NC_009085_255964_256143 /5Phos/GTATAACACTCGGCCAGCGCCAAGGTTCGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTCACACATCGCCA
CAATATGAT
clostridium_NC_013974_3097606_3097772 /5Phos/ACCATGCAGATACAATGAACCAGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGATGATAAGACACATCCAAT
TC
clostridium_FN665653_103469_103631 /5Phos/CATCAACAGCTTCTTGAAGCATTCGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTCCAACAACTATAACAGA
ACGTC
clostridium_NC_013974_117188_117346 /5Phos/AACATATCACCTGATATTCTAGTATCGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATTCCATTATATTCAAC
AGGATTGTGA
clostridium_NC_013316_3012882_3013047 /5Phos/GCTGTTGCTTGCGGATACTGGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTATATGTAGCTCAAGTTGC
clostridium_FN668375_1212250_1212413 /5Phos/AAGAGCTAATGCAGCTATTGCACTTATGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATACACTTCAGCTAT
AAGACCAT
clostridium_NC_013315_3754484_3754640 /5Phos/AACAAGAGCAGAAGTTACAGACGTGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTATAATGGTGGCTAGAGG
TGA
clostridium_FN665654_3239860_3240039 /5Phos/ACTCGTGAAGACCATGCAGATACAAGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATACTTACAATGCCTGA
GGA
clostridium_FN668941_3228320_3228491 /5Phos/ACCATGCAGATACAATGAACCGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCTGAGGATGATAAGACACATC
clostridium_NC_013974_1962664_1962825 /5Phos/GCATCTGCTGCTTCTATTGCTCCTACTGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACATGAACTGATATTA
GTTCTCCAA
clostridium_NC_003366_2769687_2769851 /5Phos/GCACAAGCTGGAGATAACATCGGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTAGAGGACGTATTCACAAT
CACT
clostridium_FN665653_127741_127918 /5Phos/CTCTATCAGCTTCTACTGCTTCTTCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCATCTCATCCACAGTTA
ATATATC
clostridium_NC_013316_2259929_2260107 /5Phos/AGATGAGATTCATACTATCGTTGGAGCTGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCAGAGAGAATAGT
AAGAGGAGA
clostridium_NC_009089_94774_94937 /5Phos/CATCAACAGCTTCTTGAAGCATTGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTCCAACAACTATAACAGAA
CG
clostridium_NC_013315_2044225_2044389 /5Phos/GTCAGCAATACGCCACCAAGCTCCTATGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTGGTGGATATCCTGT
TACC
clostridium_NC_013315_2299408_2299586 /5Phos/GCGCAATAGAGTTGTATAAGAGTGCTGGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCATTAATTATAGAT
TATAATGTATAA
clostridium_FN668941_3244255_3244408 /5Phos/GGCATAATAGGATGGATAGATGAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTAATCCAACTTCTACTGC
TAT
clostridium_NC_013316_3610909_3611065 /5Phos/GTACATTCACATATAGACCATCTTAAGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACATAGGTGCAGGTAGA
ATAGTATA
clostridium_FN665653_1104859_1105031 /5Phos/CCATACCAGTATCTTGGCATATTGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATAATGAATAACAGCAGGT
GTATTA
clostridium_NC_03366_2753681_2753838 /5Phos/AGATGAAGCACAAGCTGGAGATAAGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGGACGTATTCACAATCAC
TG
clostridium_FN665653_710906_711080 /5Phos/ATAATCATTCACCTCCATCATTCATAAGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTGAATATGGTTCGT
CTCA
clostridium_NC_009089_3706562_3706720 /5Phos/GTACATTCACATATAGACCATCTTAGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACATAGGTGCAGGTAGAA
TAGT
clostridium_NC_013316_137282_1372968 /5Phos/ACTCCACCAGGATGTTGTCCGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCGTAGGACCGTCGTGTCCAAG
clostridium_FN665652_676696_676895 /5Phos/GCAATATCAATGGTATCGAAGGCACTATGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTATTGAAGGTACTA
TTAGCGATATGC
clostridium_NC_013316_2641651_2641808 /5Phos/GTGCCGGTCTCGGTTACTCAATGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGATTATTATAATGCAGCTA
GAAG
clostridium_FN668375_3595870_3596026 /5Phos/GTACATTCACATATAGACCATCTTGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACATAGGTGCAGGTAGAAT
AGTA
clostridium_FN668941_1105700_1105868 /5Phos/AGTTCCTTCATATGACTCAGTTGATTGAGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTATATCTTCAATT
ATACATTCCTGC
clostridium_NC_013974_2505182_2505359 /5Phos/CAGCAGTTGTTGCTAGAGGTATGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCATCACCAGGTGCAGCAAGT
clostridium_NC_013315_1077126_1077298 /5Phos/GCAATTCTCTGTTGTTGTCCTCCACTCAGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGTAAGAGCCTCTTC
TTGGTCATGA
clostridium_NC_009089_2182303_2182482 /5Phos/CTATTCCTGATAATAAGTGTGTCCTCATGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGGCATCATCTAACA
ATTCTTCT
clostridium_FN665652_1909777_1909942 /5Phos/GTAATTCCAATTACTTCTAGCTCTGGTGGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTACCATCTTCTCCAT
GTGTAT
clostridium_NC_013316_3300896_3301062 /5Phos/CCATGCAGATACAATGAACCAGGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGATGATAAGACACATCCAATT
CC
clostridium_NC_013316_871338_871499 /5Phos/CCTTCTGCCATTGTAGAACAAGCTCCATGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCTGTAACTGTCCAC
TGAGC
clostridium_NC_013316_3608873_3609047 /5Phos/CAATCATGATAGAATTAGATGGAACGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCAATAGTTCCATCAGG
AGCATC
clostridium_FN665654_3717059_3717221 /5Phos/AGTGGTGAAGGTGTTCAACAAGGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTGAAGCTGGATATGTTGGAG
clostridium_NC_013315_2010489_2010657 /5Phos/CGCCTCTTCAGAAGCGGATATCAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCAGACTTCCGCCACAACCT
clostridium_NC_013315_3236301_3236474 /5Phos/GGCATAATAGGATGGATAGATGAGCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCAGCAGTTGTACCTACA
ACTAA
clostridium_NC_013315_1095924_1096090 /5Phos/AGTTCCTTCATATGACTCAGTTGATTGGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTATATCTTCAATTA
TACATTCCTGCG
enterobacter_NC_014121_4735453_4735632 /5Phos/GCATGGTAGTTCGCCAGCCGCTGGAACGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACAGCAACCGCAAGTT
CTTGACAT
enterobacter_NC_015663_1014187_1014345 /5Phos/AATATCATGGTCGTGTCCAGGCACTGGCGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTCTGGTAGCTGCT
TCTACTGTA
enterobacter_FP929040_3448334_3448513 /5Phos/AACTTACAACTACGCGCACTTGAATCGGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAGTGTTGTATGATAG
TCTCGGT
enterobacter_NC_009436_4051820_4051985 /5Phos/GCAAGTTGAGGAGATGCTGGCATGATTCGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACATGGCTCTGGAAG
ATGTGCTGATC
enterococcus_FP929058_1738439_1738606 /5Phos/GCGATAATTGTAATGATTCGTGGTGTTAGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCGTTGTCAATCCAG
TTAGTAGACT
enterococcus_CP002621_1819224_1819388 /5Phos/ACTGTGGCAGTCTATGTTCCAATTGTAGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTATCGACATAATCC
TGATAATC
enterococcus_FP929058_904007_904173 /5Phos/GCGTCGCTTCTTGCGCTCGCCGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATGTATTCATACCGTCAAGT
enterococcus_FP929058_551757_551920 /5Phos/GCCTTCACAACTACGTTGGAAGGTCTTCGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTAACAGTCCTGCCG
ACTAC
enterococcus_NC_004668_1122345_1122507 /5Phos/GCCTTCACAACTACGTTGGAAGGTCTTGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTAACAGTCCTGCCGA
CTACT
klebsiella_NC_009648_2885456_2885620 /5Phos/GCCGCTGAGCGGCGGCAAGCCGATGGCGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAATGGCAGGCCAAGC
TGAAGGCG
klebsiella_NC_009648_3899012_3899182 /5Phos/GCCAAGCGGCATTCTGGCGCCAGTGGAGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCAGACCGGAGTGGAC
AACGTCGAGGCG
klebsiella_NC_009648_4980596_4980757 /5Phos/GCCGTATATCATCGGCAATAACCGCACGGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCATGATGGTCAACA
AGGTGC
klebsiella_NC_009648_3266359_3266519 /5Phos/ACGAGCCGAGATAGGTCTGCAGCGTACGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTACTGATATTCACCA
TACTGCCG
klebsiella_NC_012731_2557467_2557634 /5Phos/GCAATATCTTCACCGGCAGCCACCGCGGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGTATATGGCACGCCA
ATCGC
klebsiella_NC_012731_4857136_4857315 /5Phos/AATAACCTTAACGTCGCCAACACGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTCGGTGAACACCTCCTGG
CACG
proteus_NC_010554_547938_548117 /5Phos/GCGGAACTGCTTGGCGTAGTAAGCGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATGTAGTGCCGTAGACCT
TCACCA
pseudomonas_NC_008463_658500_658676 /5Phos/GCGAGACCGGCGGCACCATCGTCTCCAGGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTCTGCCTGATGGAC
GTCTCCGGCTCG
pseudomonas_NC_008463_753931_754099 /5Phos/GCGGTTCACCTGTTCGCCTTCGAACACGGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCGCAGCATCTGACG
CAGGATGGTCTCG
pseudomonas_NC_009656_6431649_6431828 /5Phos/ACTCCATCGCCATCAAGGACATGGCCGGGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCGACGTGTTCCGC
ATCTTCGACGCG
pseudomonas_NC_008463_560357_560534 /5Phos/GCCTGATGCACTACAGCGCCTGGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTACCACATGGTCGATCTCGA
CGACTGC
pseudomonas_NC_010322_5224859_5225023 /5Phos/GCGCATCCAGGACGGCGAGTACGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTCGAGTGCCTGCACGAGC
TGAA
pseudomonas_NC_008463_4839746_4839924 /5Phos/GCTGGAGAACGTCAAGGTGGTGATCATCGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACCGATAACGACGAC
CGCATCAA
staph_FN433596_2844085_2844263 /5Phos/ACGATTGGAGAAGGCAGTGTGATTGGGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGACAGATTACAATTGG
CG
staph_NC_009632_1198350_1198529 /5Phos/GCCGCAATACCGATATTCCAGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCCCATTGTCCACCAGCTGAACCG
staph_FN433596_2521244_2521419 /5Phos/GTGAAGGTCGTGCTCCTATCGGTGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGATCTGGTGAAGTTCGTAT
GAT
staph_NC_009487_430842_431017 /5Phos/GCTGGTACTTGTACTTATATCGAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCAGAAGATGATATCGTTA
CGTCAT
staph_NC_009782_2086681_2086849 /5Phos/GCGCATATTGCATTAATGGCTATAGATGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCAGCAGGTTATACA
CTCG
staph_NC_009782_58256_58423 /5Phos/GCAATTCTTACCACAGCACGAAGAACAGGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTAGATGAAGATA
ATGAAGTCG
staph_NC_013450_991049_991222 /5Phos/GCATCTTCATACAATACTTCTAGCTTACGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACAATACCAGTTGT
ATTACG
staph_NC_013450_1360842_1361008 /5Phos/GCTTCAGCGCCATTACCGCCACCAGCTGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTCTTGATATATTCT
TGTAAGCG
staph_AM990992_2526026_2526192 /5Phos/GTTCACACAACGCGCCGACTAGAATCCGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACGATATCCAAGATA
ATGATTGGCTA
staph_NC_010079_361284_361447 /5Phos/GCGCACCTACAATCGCCATTACTACACGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACTCATTATCGACTGT
TACATCGACTGA
staph_NC_007795_2085723_2085901 /5Phos/AGCGCACATGTGACAGCGTGTAGGTTAGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTGCCTTAGATTGTTC
AGAACAAT
staph_NC_009641_23125_23297 /5Phos/CGAATGGATATGTACCATGGTCGATATCGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTCTCTAATATGATG
TCCAT
staph_FN433596_2144570_2144734 /5Phos/ACTACAACAGCAACCGCATTACAATGGCGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGTGCTAAGAGGTCA
TCGGA
staph_NC_009782_54857_55020 /5Phos/AGCTTCAGATAAGTACCTATCTGAGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGAAGAATAGTTATTCTTG
ATAATGTAT
staph_AM990992_1656616_1656789 /5Phos/CGTATTGCTCGAATACATGATAGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCACAATGTATCAAGGCCAGCT
staph_NC_007793_44227_44395 /5Phos/GCGACCAGTTGTTATCGACCGTGTGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCAGAACGATACGGTGCTGT
ATA
staph_NC_009641_1102949_1103116 /5Phos/CAATTACATTGTCTGTTGCGTAGATACCGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTGTGGCTAATGTG
CCAGTT
staph_NC_009641_1137731_1137898 /5Phos/GCACCACTCTATAGCAGTAGCGTATTGGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACAGCCAATGTCACCT
AAGTCAACA
staph_FN433596_2715713_2715871 /5Phos/ACAGTCCGAATAAGATACGACTATTCGAGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTTGTAACGTATAT
GAATAGTTGA
staph_NC_009782_606652_606825 /5Phos/AGATGCAATAACAGGTCGAATATTAATTGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCATAGTGAGAGTA
GTGAA
staph_FN433596_657625_657803 /5Phos/AGATGCAATAACAGGTCGAATATTAAGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACACATACGGCCATAGT
GAGAG
species_NC_004741_4338803_4338982 /5Phos/GAACATAACGCGACGTTCCAGCTGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTTCAGAGGTGTTGTAGT
CG
species_NC_009648_4535521_4535683 /5Phos/GCGCTGGCGCAGTATCGTGAACTGGGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACCAACGTAATCTCTATT
ACCG
species_NC_010410_3677607_3677782 /5Phos/GCTGTAATGCAAGTAGCGTATGCGCTCAGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAGGCCGCCAATGCC
TGACG
species_CP001844_589057_589217 /5Phos/GCCTGTAGCAACAGTACCACGACCAGTGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACCACGTAATAATGC
ACCAA
species_CP002110_2761329_2761492 /5Phos/ACTACGCTGAAGCTGGTGACAACATTGGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTTGAGGACGTATTCT
CAATC
species_NC_010473_3546640_3546818 /5Phos/GCTGGTACTTACGTTCAGATGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCACGGTGAACGCCGTTACATCC
species_CP001844_57304_57465 /5Phos/GCAATTCTTACCACAGCACGAAGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTAGATGAAGATAATGAAG
TCG
species_NC_012731_1975396_1975559 /5Phos/GCGGCGGCAGGCGGTAACGCCAGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGCGGTTATCTACCACGGCG
species_NC_003923_198857_199024 /5Phos/GCACCTACTTGTCCAGCACCAGCCATGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATACCACCACCAATAC
AAGCA
species_NC_010400_52102_52263 /5Phos/GCGCGGTAACATGCCATATTCTGCGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCCTGAATGACATCACAGTCG
species_NC_010473_3310005_3310164 /5Phos/AATCAGGTCAAGGAACTGCAAGCGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTCTCAATCATATGCACCGG
AATAC
species_FP929058_3022053_3022226 /5Phos/GAACATATGTGTATGACGATGCGCGGGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTACATGTCGCTTATCT
GCCAGAAGGT
species_NC_009085_1010393_1010556 /5Phos/CGTGTGCGTAGTGACGAGTTGGAGAGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGAATACGATGATGTAAG
GTACACCTA
species_CP002621_172633_172802 /5Phos/CAGGAGTTACTTCTGTTCCATGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTGAACAATTAGATCACCTCG
species_FP929040_442484_442653 /5Phos/CGTAATCTCCATTACCGATGGTCAGATCGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCACGTATTCTACCTCC
ACTCTCGTCT
species_NC_003923_1334345_1334501 /5Phos/CATTCGACGTTCTGGTATTACTTGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACGCTCCGCATCAGCAGCA
CCACGTT
species_NC_009085_1010678_1010853 /5Phos/CTGAACCACGGATTACTGGAGTGTCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCCTGTTACTACTGTACC
ACGAC
pseudomonas_NC_008463_4756080_4756240 /5Phos/GAATCGAACGGTCTCATTAACAGATGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTTTCCAGGGATATAAG
ACGC
pseudomonas_NC_002516_1063894_1064077 /5Phos/CCCGCAGAGTCACACTCGGAGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCACTCTTGGTACTACTCACTAGC
pseudomonas_NC_008463_3182693_3182865 /5Phos/GAGTCTCTTTCAACCTGGATTAGATATGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAAGATTAATAGCGTAC
TTTACTCC
pseudomonas_NC_009656_2819490_2819655 /5Phos/ATCCCGCAGATACTAGGTTCTTAATGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAACTATTCATATTACAC
CCTAAGG
pseudomonas_NC_008463_3184022_3184185 /5Phos/CAGTGGGCTATCCTAAGCCAAAGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATAAGCGAACTAACTATCA
CTTA
pseudomonas_NC_002516_1065937_1066093 /5Phos/ACAAAGCGTTCTAAACGATTAGAACTGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGAGAAAGGAAACAGGA
TAGTAC
pseudomonas_NC_002516_1067833_1068007 /5Phos/CCAATGGAGAAGTCTAAATGTCCAAGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTATCAGAGATACATGAC
TCTTAGG
pseudomonas_NC_008463_3182351_3182508 /5Phos/CGAATCACTGGACTACATTTATATTTCTGTTGGAGGCTCATCGTTCCTAT
ATTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAGCGAACCTTTATAT
TTGACCAT
pseudomonas_NC_008463_3184314_3184473 /5Phos/CTCAAGTCTTGCCCTGATAGAATTATGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCACGACTTATCTACTT
TAGAAATC
pseudomonas_AP012280_3765216_3765383 /5Phos/GGTGATCGTTATTATGATAGTACGGCGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTCGGTTAAGGGAATTA
CGAC
pseudomonas_AP012280_3765033_3765192 /5Phos/ACTCGGATGGTAGGTTTATTAAAGCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTGATCGTTATTATGATA
GTACGG
enterococcus_NZ_GG703715_13422_13573 /5Phos/ACAATCGTTGTCGCACTGCATAGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAACTTGGTCTACCGTACCAC
enterococcus_NZ_GG703582_76982_77140 /5Phos/GGATAATACAATCCTAATACGTACGGAGTTGGAGGCTCATCGTTCCTATA
TTCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTGCTGTAACTAGGG
TAGC
enterococcus_NZ_GL455004_28219_28381 /5Phos/CTATATTCAACGGGTCACGGGTAGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCATTGATTCGATCTCGTA
ACTC
enterococcus_NZ_GG703720_94699_94852 /5Phos/AATGTTATTGTGGTTGCGTGTTCGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTACTTTGGAAGTGCCCTGAC
enterococcus_NZ_GG703715_15795_15951 /5Phos/CATGTCTTCTAGTACAGGTTTGCCGGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTAAGAGGCCGCTAACT
TC
enterococcus_NZ_GL455899_32848_32984 /5Phos/CTCTGGCTCGTGGGCTCGGGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCTTCTTGAGATAGTCCGGTATAATC
enterococcus_NZ_GG692918_325104_325257 /5Phos/ATTCGATCACGATGGGCTGGGGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCAATTTCCTGTGTCATACACGC
enterococcus_NC_004668_920608_920750 /5Phos/CAATTGATTTAGCCACTACACCTTACGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCACTATTCTGGCGACCA
CC
enterococcus_NZ_GG703575_78829_78963 /5Phos/GATAAAGAAGCGTCTTGACCCAGTGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCATCTGGTGCTCCTTGACGC
enterococcus_NZ_GL455931_26355_26493 /5Phos/GCAAATTTAGAGAGTGCATGCATGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGGAAGAGGACGGCATACAAC
enterococcus_NZ_GG669058_207026_207172 /5Phos/CATTTCATCTAGACCGCTCGTGTGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCTTGAAGTGTATGTTGGGAC
proteus_NZ_GG661998_111187_111342 /5Phos/GTCGCCCTCGTGCTAACGTGTTGGAGGCTCATCGTTCCTATATTCCACAC
CACTTATTATTACAGATGTTATGCTCGCAGGTCGGTTCTTTGATGTACCGGTT
proteus_NC_010554_2037943_2038091 /5Phos/GCTGATGACGGTGAAGTTTATCAGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCATTATCGCACATATTGACC
AC
proteus_NZ_GG668576_810893_811054 /5Phos/GAAATTAGCTAAAGGGATATCGCGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCAACTTTCCGCCAATCCTGC
proteus_NZ_GG668594_760_939 /5Phos/CACCTACGTTCTCACCTGCACGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCATTCGATAGTACCAGTTACGTC
proteus_NZ_GG668579_22072_22234 /5Phos/GTTGCTTATAGCGTCGCTGCTGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTGGTTATCGAGAAGATAAAGG
proteus_NC_010554_2448957_2449119 /5Phos/GTAAGCGTAGCGATACGTTGAGGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAGTGAACGCACCACTGG
proteus_NC_010554_3033758_3033936 /5Phos/TCAGGTAGAGAATACTCAGGCGCGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGGAGAAGGCTAGGTTGTC
proteus_NC_010554_454391_454540 /5Phos/GCAACCCACTCCCATGGTGTGTTGGAGGCTCATCGTTCCTATATTCCACA
CCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTTCTTCATCAGACAATCTG
gyrB_NC_015663_1455472_1455621 /5Phos/GCCCTTTCAGGACTTTGATACTGGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTACGGAGACGGAGTTAT
CG
gyrB_NC_010410_4215_4366 /5Phos/ACACTGACCGATTCATCCTCGTGGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTGAAAGTGCGTTAACAACC
gyrB_NC_005773_4904_5052 /5Phos/CGGAAGCCCACCAAGTGAGTACGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGAAACCAGTTTGTCCTTAGTC
gyrB_NC_016514_5343_5487 /5Phos/ACCAGCTTGTCTTTAGTCTGAGAGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTTACGACGGGTCATTTC
AC
gyrB_NC_016603_2631439_2631616 /5Phos/CATTGGTTTGTTCTGTTTGAGAGGCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGATTCATCTTCGTGAATT
GTGAC
gyrB_NC_009436_4366_4524 /5Phos/GGACTTTGATACTGGAGGAGTCATAGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTGTACGGAAACGGAGTTA
TCG
gyrB_NC_009512_4203_4373 /5Phos/ATGCTGGAGGAGTCGTACGTTTGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTCGCGCACACTAATAGATTC
pseudomonas_NC_009085_307050_307218 /5Phos/AACTAAACCTACACGGAATTGGTTCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGCAGATACACGACGTTTA
TGT
pseudomonas_NC_009085_308225_308377 /5Phos/GCCGCTTCACCTACGTTAGGAAGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGTAAAGATGAGTCTTTAACG
TC
pseudomonas_NC_016612_1674334_1674490 /5Phos/GACGTTTGTGCGTAATCTCAGACGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAGGAAACCGTATTCGTTCGT
pseudomonas_NC_016603_3425179_3425337 /5Phos/ACAACACTTTACCACTTGAGTGGGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGTAACTGCCCATGTCAAGA
TAC
pseudomonas_NC_016603_3427629_3427808 /5Phos/CCACGTTTAGTTGAACCACCGCGTTGGAGGCTCATCGTTCCTATATTCCA
CACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCAATACGCCAGTTGTTAGTTC
pseudomonas_NC_010410_3543925_3544088 /5Phos/AATCGATAATAAGTACGGTGCATCCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGAAGAATACATTCGCGTA
CATC
pseudomonas_NC_005966_304936_305079 /5Phos/AAGCAAGATCGAGTCTTCATAGTTGGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGATATACACGATACCTGA
TTCGT
pseudomonas_NC_008593_226005_226171 /5Phos/CCGATATTCATACGAGAAGGTACACGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCAGTAACTCTATTGTCAA
ACGGT
pseudomonas_NC_016514_213592_213738 /5Phos/GTAGTGAGTCGGGTGTACGTCTCGTTGGAGGCTCATCGTTCCTATATTCC
ACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCTTCGATAGCAGACAGATA
GT
pseudomonas_NC_005966_303883_304054 /5Phos/ACCTACACGGAATTGGTTCTCAGTGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGATACACGACGTTTGTGTG
TA
enterobacter_NC_014618_3997909_3998085 /5Phos/CAACATCATTAGCTTGGTCGTGGGGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCTTGCGTGTTACCAACTCGTC
enterobacter_NZ_GL892086_61549_615324 /5Phos/CGGCACGTCCGAATCGTATCAGTTGGAGGCTCATCGTTCCTATATTCCAC
ACCACTTATTATTACAGATGTTATGCTCGCAGGTCTCGTGTCCCGTATATGTTGG
enterobacter_NZ_GL892086_1664663_1664834 /5Phos/AATAGAGGCCCACAAGTCTTGTTCGTTGGAGGCTCATCGTTCCTATATTC
CACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCGCTCTCCACTATGGGTAGT
enterobacter_NZ_GG704865_427821_427978 /5Phos/GCTACATTAATCACTATGGACAGACAGTTGGAGGCTCATCGTTCCTATAT
TCCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCGATGGTCGATCTATCGT
CTCT
enterobacter_NZ_GL892087_1610708_1610874 /5Phos/GAAGTGTTATTCAAACTTTGGTCCCGTTGGAGGCTCATCGTTCCTATATT
CCACACCACTTATTATTACAGATGTTATGCTCGCAGGTCCTTGAACCCTTGGTTCAA
GGT
TABLE 2
A list of organisms for which the methods and kits described herein have been
validated to detect using the compositions described herein
Acinetobacter baumannii 1656-2
Acinetobacter baumannii AB0057
Acinetobacter baumannii AB307-0294
Acinetobacter baumannii ACICU
Acinetobacter baumannii ATCC 17978
Acinetobacter baumannii AYE
Acinetobacter baumannii MDR-ZJ06
Acinetobacter baumannii SDF
Acinetobacter baumannii TCDC-AB0715
Acinetobacter calcoaceticus PHEA-2
Acinetobacter sp. ADP1
Acinetobacter sp. DR1
Clostridium acetobutylicum ATCC 824
Clostridium acetobutylicum DSM 1731
Clostridium acetobutylicum EA 2018
Clostridium beijerinckii NCIMB 8052
Clostridium botulinum A2 str. Kyoto
Clostridium botulinum A3 str. Loch Maree
Clostridium botulinum A str. ATCC 19397
Clostridium botulinum A str. ATCC 3502
Clostridium botulinum A str. Hall
Clostridium botulinum B1 str. Okra
Clostridium botulinum Ba4 str. 657
Clostridium botulinum BKT015925
Clostridium botulinum B str. Eklund 17B
Clostridium botulinum E3 str. Alaska E43
Clostridium botulinum F str. 230613
Clostridium botulinum F str. Langeland
Clostridium botulinum H04402 065
Clostridium cellulolyticum H10
Clostridium cellulovorans 743B
Clostridium clariflavum DSM 19732
Clostridium difficile 630
Clostridium difficile BI1
Clostridium difficile BI9
Clostridium difficile CD196
Clostridium difficile strain 2007855
Clostridium difficile strain CF5
Clostridium difficile strain M120
Clostridium difficile M68
Clostridium difficile R20291
Clostridium kluyveri DSM 555
Clostridium kluyveri NBRC 12016
Clostridium lentocellum DSM 5427
Clostridium ljungdahlii DSM 13528
Clostridium novyi NT
Clostridium perfringens ATCC 13124
Clostridium perfringens SM101
Clostridium perfringens str. 13
Clostridium phytofermentans ISDg
Clostridium saccharolyticum-like K10
Clostridium saccharolyticum WM1
Clostridium sp. SY8519
Clostridium sticklandii DSM 519
Clostridium tetani E88
Clostridium thermocellum ATCC 27405
Clostridium thermocellum DSM 1313
Enterobacter aerogenes KCTC 2190
Enterobacter asburiae LF7a
Enterobacter cloacae SCF1
Enterobacter cloacae subsp.cloacae ATCC 13047
Enterobacter cloacae subsp. cloacae NCTC 9394
Enterobacter sp. 638
Enterococcus faecalis 62
Enterococcus faecalis OG1RF
Enterococcus faecalis V583
Enterococcus sp. 7L76
Escherichia coli 042
Escherichia coli 536
Escherichia coli 55989
Escherichia coli ABU 83972
Escherichia coli APEC O1
Escherichia coli ATCC 8739
Escherichia coli BL21(DE3)
Escherichia coli ‘BL21-Gold(DE3)pLysS AG'
Escherichia coli B str. REL606
Escherichia coli BW2952
Escherichia coli CFT073
Escherichia coli DH1 (ME8569)
Escherichia coli E24377A
Escherichia coli ED1a
Escherichia coli ETEC H10407
Escherichia coli HS
Escherichia coli IAI1
Escherichia coli IAI39
Escherichia coli IHE3034
Escherichia coli KO11
Escherichia coli LF82
Escherichia coli NA114
Escherichia coli O103: H2 str. 12009
Escherichia coli O111:H-str. 11128
Escherichia coli O127:H6 str. E2348/69
Escherichia coli O157:H7 str. EC4115
Escherichia coli O157:H7 str. EDL933
Escherichia coli O157:H7 str. Sakai
Escherichia coli O157:H7 str. TW14359
Escherichia coli O26:H11 str. 11368
Escherichia coli O55:H7 str. CB9615
Escherichia coli O7:K1 str. CE10
Escherichia coli O83:H1 str. NRG 857C
Escherichia coli S88
Escherichia coli SE11
Escherichia coli SE15
Escherichia coli SMS-3-5
Escherichia coli str. ‘clone D i14’
Escherichia coli str. ‘clone D i2’
Escherichia coli str. K-12 substr. DH10B
Escherichia coli str. K-12 substr. MDS42
Escherichia coli str. K-12 substr. MG1655
Escherichia coli str. K12 substr. W3110
Escherichia coli UM146
Escherichia coli UMN026
Escherichia coli UMNK88
Escherichia coli UTI89
Escherichia coli W
Escherichia fergusonii ATCC 35469
Klebsiella pneumoniae 342
Klebsiella pneumoniae KCTC 2242
Klebsiella pneumoniae NTUH-K2044
Klebsiella pneumoniae subsp. pneumoniae MGH
78578
Klebsiella variicola At-22
Proteus mirabilis HI4320
Pseudomonas aeruginosa LESB58
Pseudomonas aeruginosa M18
Pseudomonas aeruginosa NCGM2.S1
Pseudomonas aeruginosa PA7
Pseudomonas aeruginosa PAO1
Pseudomonas aeruginosa UCBPP-PA14
Pseudomonas brassicacearum subsp. brassicacearum
NFM421
Pseudomonas entomophila L48
Pseudomonas fluorescens F113
Pseudomonas fluorescens Pf0-1
Pseudomonas fluorescens Pf-5
Pseudomonas fluorescens SBW25
Pseudomonas fulva 12-X
Pseudomonas mendocina NK-01
Pseudomonas mendocina ymp
Pseudomonas putida BIRD-1
Pseudomonas putida F1
Pseudomonas putida F1
Pseudomonas putida GB-1
Pseudomonas putida KT2440
Pseudomonas putida S16
Pseudomonas putida W619
Pseudomonas stutzeri A1501
Pseudomonas stutzeri ATCC 17588 = LMG 11199
Pseudomonas stutzeri DSM 4166
Pseudomonas syringae pv. phaseolicola 1448A
Pseudomonas syringae pv. syringae B728a
Pseudomonas syringae pv. tomato str. DC3000
Shigella boydii CDC 3083-94
Shigella boydii Sb227
Shigella dysenteriae Sd197
Shigella flexneri 2002017
Shigella flexneri 2a str. 2457T
Shigella flexneri 2a str. 301
Shigella flexneri 5 str. 8401
Shigella sonnei Ss046
Staphylococcus aureus
Staphylococcus carnosus subsp. carnosus
Staphylococcus epidermidis
Staphylococcus haemolyticus JCSC1435
Staphylococcus lugdunensis
Staphylococcus pseudintermedius
Staphylococcus saprophyticus subsp.
Staphylococcus aureus
Staphylococcus saprophyticus
Staphylococcus epidermis
Acinetobacter baumannii
Enterococcus faecalis
Enterobacter cloacae
Enterobacter aerogenes
Enterococcus faecium
Candida albicans
Klebsiella pneumoniae
Escherichia coli
Clostridium difficile
Proteus mirabilis
Pseudomonas aeruginosa
TABLE 3
Genus level regions can be used for coarse discrimination of organisms.
Probe Coordinates Gene
species_NC_004741_43388 rpsC, S4416, 30S ribosomal protein S3
03_4338982
species_NC_009648_45355 atpA, KPN_04139, F0F1 ATP synthase subunit alpha
21_4535683
species_NC_010410_36776 int, ABAYE3575, integrase/recombinase (E2 protein)
07_3677782
species_CP001844_589057_ tufA, SA2981_0525, Translation elongation factor Tu
589217
species_CP002110_276132 tuf, HMPREF0772_12641, elongation factor EF1A
9_2761492
species_NC_010473_35466 rp1B, ECDH10B_3492, 50S ribosomal protein L2
40_3546818
species_CP001844_57304_ tnpB, SA2981_0055, Transposase B from transposon
57465 Tn554 tnpB, SA2981_1617, Transposase B from
transposon Tn554
species_NC_012731_19753 putA, KP1_2030, trifunctional transcriptional
96_1975559 regulator/proline dehydrogenase/pyrroline-5-
carboxylate dehydrogenase
species_NC_003923_19885 MW0166, hypothetical protein
7_199024
species_NC_010400_52102_ rph, ABSDF0051, ribonuclease PH
52263
species_NC_010473_33100 rpoD, ECDH10B_3242, RNA polymerase sigma factor RpoD
05_3310164
species_FP929058_302205 ENT_30090, GTP cyclohydrolase I
3_3022226
species_NC_009085_10103 A1S_0279, elongation factor Tu
93_1010556
species_CP002621_172633_ rplO, OG1RF_10170, 50S ribosomal protein L15
172802
species_FP929040_442484_ ENC_04200, proton translocating ATP synthase, F1
442653 alpha subunit
species N0_003923_13343 katA, MW1221, catalase
45_133401
species_NC 009085_10106 A1S_0279, elongation factor Tu
78_101053
TABLE 4
Genus level probes
Probe Coordinates Binding region 1 Binding region 2
species_NC_004741_4338803_4338982 GAACATAACGCGACGTTCCAGCTG GCTTCAGAGGTGTTGTAGTCG
species_NC_009648_4535521_4535683 GCGCTGGCGCAGTATCGTGAACTGG ACCAACGTAATCTCTATTACCG
species_NC_010410_3677607_3677782 GCTGTAATGCAAGTAGCGTATGCGCTCA AAGGCCGCCAATGCCTGACG
species_CP001844_589057_589217 GCCTGTAGCAACAGTACCACGACCAGT CACCACGTAATAATGCACCAA
species_CP002110_2761329_2761492 ACTACGCTGAAGCTGGTGACAACATTG GTTGAGGACGTATTCTCAATC
species_NC_010473_3546640_3546818 GCTGGTACTTACGTTCAGAT ACGGTGAACGCCGTTACATCC
species_CP001844_57304_57465 GCAATTCTTACCACAGCACGAA ATCTAGATGAAGATAATGAAGTCG
species_NC_012731_1975396_1975559 GCGGCGGCAGGCGGTAACGCCAG ACGCGGTTATCTACCACGGCG
species_NC_003923_198857_199024 GCACCTACTTGTCCAGCACCAGCCAT AATACCACCACCAATACAAGCA
species_NC_010400_52102_52263 GCGCGGTAACATGCCATATTCTGC CCTGAATGACATCACAGTCG
species_NC_010473_3310005_3310164 AATCAGGTCAAGGAACTGCAAGC GTCTCAATCATATGCACCGGAATAC
species_FP929058_3022053_3022226 GAACATATGTGTATGACGATGCGCGG GTACATGTCGCTTATCTGCCAGAAG
GT
species_NC_009085_1010393_1010556 CGTGTGCGTAGTGACGAGTTGGAGA AGAATACGATGATGTAAGGTACACC
TA
species_CP002621_172633_172802 CAGGAGTTACTTCTGTTCCAT TTGAACAATTAGATCACCTCG
species_FP929040_442484_442653 CGTAATCTCCATTACCGATGGTCAGATCC ACGTATTCTACCTCCACTCTCGTCT
species_NC_003923_1334345_1334501 CATTCGACGTTCTGGTATTACTT CACGCTCCGCATCAGCAGCACCACG
TT
species_NC_009085_1010678_1010853 CTGAACCACGGATTACTGGAGTGTC GCCTGTTACTACTGTACCACGAC
TABLE 5
Species/strain level regions can be used for discrimination at the level of
species and strains.
Probe Coordinates Gene
acinetobacter_NC_010 ACICU_00572, pyridine nucleotide transhydrogenase
611_627997_628164 (proton pump) subunit alpha (part2)
acinetobacter_NC_010 pepN, ACICU_02288, aminopeptidase N
611_2417580_2417755 trpC, ACICU_02557, indole-3-glycerol-phosphate
synthase
acinetobacter_CP0025 recF, ABTW07_0010, recombination protein F
22_11753_11931
acinetobacter_NC_011 gshB, AB57_3788, glutathione synthetase
586_3908329_3908508
acinetobacter_NC_010 ACICU_00129, NAD-dependent aldehyde dehydrogenase
611_145181_145340
acinetobacter_NC_010 ACICU_03630, A/G-specific DNA glycosylase
611_3854494_3854662
acinetobacter_NC_010 nadC, ABSDF0056, nicotinate-nucleotide
400_56216_56383 pyrophosphorylase (quinolinate
phosphoribosyltransferase)
acinetobacter_NC_010 near ACICU_01347, carbonic anhydrase
611_1454960_1455136
acinetobacter_NC_009 A1S_0230, phosphoglyceromutase
085_255964_256143
clostridium_NC_01397
4_3097606_3097772
clostridium_FN665653_
103469_103631
clostridium_NC_01397
4_117188_117346
clostridium_NC_01331 nifJ, CDR20291_2570, pyruvate-flavodoxin
6_3012882_3013047 oxidoreductase
clostridium_FN668375_
1212250_1212413
clostridium_NC_01331 pykF, CD196_3170, pyruvate kinase
5_3754484_3754640
clostridium_FN665654_
3239860_3240039
clostridium_FN668941_
3228320_3228491
clostridium_NC_01397
4_1962664_1962825
clostridium_NC_00336 tuf, CPE2407, elongation factor Tu
6_2769687_2769851
clostridium_FN665653_
127741_127918
clostridium_NC_01331 clpB, CDR20291_1933, chaperone
6_2259929_2260107
clostridium_NC_00908 rpoC, CD0067, DNA-directed RNA polymerase subunit
9_94774_94937 beta'
clostridium_NC_01331 CD196_1764, cell surface protein
5_2044225_2044389
clostridium_NC_01331 near CD196_1987, multiprotein-complex assembly
5_2299408_2299586 protein
clostridium_FN668941_
3244255_3244408
clostridium_NC_01331 gpmI, CDR20291_3027, phosphoglyceromutase
6_3610909_3611065
clostridium_FN665653_
1104859_1105031
clostridium_NC_00336 tuf, CPE2407, elongation factor Tu
6_2753681_2753838
clostridium_FN665653_
710906_711080
clostridium_NC_00908 gpmI, CD3171, phosphoglyceromutase
9_3706562_3706720
clostridium_NC_01331 dnaF, CDR20291_1146, DNA polymerase III PolC-type
6_1372812_1372968
clostridium_FN665652_
676696_676895
clostridium_NC_01331 CDR20291_2249
6_2641651_2641808
clostridium_FN668375_
3595870_3596026
clostridium_FN668941_
1105700_1105868
clostridium_NC_01397
4_2505182_2505359
clostridium_NC_01331 potA, CD196_0900, spermidine/putrescine ABC
5_1077126_1077298 transporter ATP-binding protein
clostridium_NC_00908 CD1878A
9_2182303_2182482
clostridium_FN665652_
1909777_1909942
clostridium_NC_01331 ntpB, CDR20291_2788, V-type ATP synthase subunit B
6_3300896_3301062
clostridium_NC_01331 spoVAD, CDR20291_0703, stage V sporulation protein AD
6_871338_871499
clostridium_NC_01331 bclA2, CDR20291_3090, exosporium glycoprotein
6_3608873_3609047 eno, CDR20291_3026, enolase
clostridium_FN665654_
3717059_3717221
clostridium_NC_01331 CD196_1739, hypothetical protein
5_2010489_2010657
clostridium_NC_01331 adhE, CD196_2753, bifunctional acetaldehyde-
5_3236301_3236474 CoA/alcohol dehydrogenase CD196_2095, sodium:solute
symporter spoVD, CD196_2497, stage V sporulation
protein D (sporulation specific penicillin-binding
protein)
clostridium_NC_01331 CD196_0911, N-acetylmuramoyl-L-alanine amidase
5_1095924_1096090
enterobacter_NC_0141 ECL_04612, 50S ribosomal subunit protein L13
21_4735453_4735632
enterobacter_NC_0156 EAE_24795, hemagluttinin domain-containing
63_1014187_1014345 protein, rp1R, EAE_04875, 50S ribosomal protein L18
enterobacter_FP92904
0_3448334_3448513
enterobacter_NC_0094 rplD, Ent638_3750, 50S ribosomal protein L4
36_4051820_4051985
enterococcus_FP92905 ENT_17660, hypothetical protein
8_1738439_1738606
enterococcus_CP00262 OG1RF_11736, group 2 glycosyl transferase
1_1819224_1819388
enterococcus_FP92905 near ENT_09350, Uncharacterized protein conserved in
8_904007_904173 bacteria
enterococcus_FP92905
8_551757_551920
enterococcus_NC_0046
68_1122345_1122507
klebsiella_NC_009648_ mqo, KPN_02629, malate:quinone oxidoreductase
2885456_2885620
klebsiella_NC_009648_ garL, KPN_03538, alpha-dehydro-beta-deoxy-D-glucarate
3899012_3899182 aldolase
klebsiella_NC_009648_ frdB, KPN_04552, fumarate reductase iron-sulfur
4980596_4980757 subunit
klebsiella_NC_009648 KPN_02970, integral transmembrane protein; acridine
3266359_3266519 resistance
klebsiella_NC_012731_ KP1_2672, putative malate dehydrogenase
2557467_2557634
klebsiella_NC_012731_ glpR, KP1_5123, DNA-binding transcriptional repressor
4857136_4857315 GlpR
proteus_NC_010554_54 PMI0497, phage terminase large subunit
7938_548117
pseudomonas_NC_00846 PA14_07660, hypothetical protein
3_658500_658676
pseudomonas_NC_00846 rpoC, PA14_08780, DNA-directed RNA polymerase subunit
3_753931_754099 beta'
pseudomonas_NC_00965 oadA, PSPA7_6223, pyruvate carboxylase subunit B
6_6431649_6431828
pseudomonas_NC_00846 PA14_06330, serine/threonine protein kinase
3_560357_560534
pseudomonas_NC_01032 PputGB1_0612, arginine decarboxylase
2_5224859_5225023 PputGB1_4676, ketol-acid reductoisomerase
pseudomonas_NC_00846 dadA, PA14_70040, D-amino acid dehydrogenase small
3_4839746_483924 subunit ung, PA14_54590, uracil-DNA glycosylase
staph_FN433596_28440 SATW20_26770, putative acetyltransferase
85_2844263
staph_NC_009632_1198 SaurJH1_1177, branched-chain alpha-keto acid
350_1198529 dehydrogenase subunit E2
staph_FN433596_25212 rplB, SATW20_23810, 50S ribosomal protein L2
44_2521419
staph_NC_009487_4308 SaurJH9_0396, hypothetical protein
42_431017
staph_NC_009782_2086 SAHV_1928, truncated amidase
681_2086849
staph_NC_009782_5825 tnpB, SAHV_1645, transposase B
6_58423
staph_NC_013450_9910 SAAV_0970, ribosomal large subunit pseudouridine
49_991222 synthase D
staph_NC_013450_1360 opuD1, SAAV_1329, BCCT family osmoprotectant
842_1361008 transporter
staph_AM990992_25260 SAPIG2450, nitrate reductase, alpha subunit
26_2526192
staph_NC 010079_3612 near USA300HOU_0330, PfoR family transcriptional
84_361447 regulator
staph_NC_007795_2085 SAOUHSC_02251, hypothetical protein
723_2085901
staph_NC_009641_2312 purA, NWMN_0016, adenylosuccinate synthetase
5_23297
staph_FN433596_21445 hlb, SATW20_19320, phospholipase C precursor
70_2144734 (pseudogene), SATW20_19830, phage protein
staph_NC_009782_5485 SAHV_0049, hypothetical protein
7_55020
staph_AM990992_16566 proC, SAPIG1569, pyrroline-5-carboxylate reductase
16_1656789
staph_NC_007793_4422 SAUSA300_0036, hypothetical protein
7_44395
staph_NC_009641_1102 NWMN_0995, phage anti-repressor protein
949_1103116
staph_NC_009641_1137 NWMN_0310, phage tail fiber
731_1137898
staph_FN433596_27157 SATW20_25670, putative amino acid permease
13_2715871
staph_NC_009782_6066 rpoB, SAHV_0540, DNA-directed RNA polymerase subunit
52_606825 beta
staph_FN433596_65762 rpoB, SATW20_06120, DNA-directed RNA polymerase beta
5_657803 chain protein
pseudomonas_NC_00846
3_4756080_4756240
pseudomonas_NC_00251
6_1063894_1064077
pseudomonas_NC_00846 PA14_35780, hypothetical protein
3_3182693_3182865
pseudomonas_NC_00965 PSPA7_0044, filamentous hemagglutinin
6_2819490_2819655
pseudomonas_NC_00846 PA14_35790, homospermidine synthase
3_3184022_3184185
pseudomonas_NC_00251 PA0984, colicin immunity protein
6_1065937_1066093
pseudomonas_NC_00251 near pyoS5, PA0985, pyocin S5
6_1067833_1068007
pseudomonas_NC_00846 PA14_35780, hypothetical protein
3_3182351_3182508
pseudomonas_NC_00846 PA14_35790, homospermidine synthase
3_3184314_3184473
pseudomonas_AP012280_
3765216_3765383
pseudomonas_AP012280_
3765033_3765192
enterococcus_NZ_GG70
3715_13422_13573
enterococcus_NZ_GG70
3582_76982_77140
enterococcus_NZ_GL45
5004_28219_28381
enterococcus_NZ_GG70
3720_94699_94852
enterococcus_NZ_GG70
3715_15795_15951
enterococcus_NZ_GL45
5899_32848_32984
enterococcus_NZ_GG69
2918_325104_325257
enterococcus_NC_0046 EF0957, maltose phosphorylase
68_920608_920750
enterococcus_NZ_GG70
3575_78829_78963
enterococcus_NZ_GL45
5931_26355_26493
enterococcus_NZ_GG66
9058_207026_207172
proteus_NZ_GG661998_
111187_111342
proteus_NC_010554_20 lepA, PMI1890, GTP-binding protein LepA
37943_2038091
proteus_NZ_GG668576_
810893_811054
proteus_NZ_GG668594_
760_939
proteus_NZ_GG668579_
22072_22234
proteus_NC_010554_24 PMIr002
48957_2449119
proteus_NC_010554_30 PMIr002
33758_3033936
proteus_NC_010554_45 PMIr006
4391_454540
pseudomonas_NC_00908 rpoB, A1S_0287, DNA-directed RNA polymerase subunit
5_307050_307218 beta
pseudomonas_NC_00908 rpoB, A1S_0287, DNA-directed RNA polymerase subunit
5_308225_308377 beta
pseudomonas_NC_01661 rpoB, KOX_07910, DNA-directed RNA polymerase subunit
2_1674334_1674490 beta
pseudomonas_NC_01660 rpoB, BDGL_003192, RNA polymerase subunit B
3_3425179_3425337
pseudomonas_NC_01660 rpoB, BDGL_003193, DNA-directed RNA polymerase subunit
3_3427629_3427808 beta
pseudomonas_NC_01041 rpoB, ABAYE3489, DNA-directed RNA polymerase subunit
0_3543925_3544088 beta
pseudomonas_NC_00596 rpoB, ACIAD0307, DNA-directed RNA polymerase subunit
6_304936_305079 beta
pseudomonas_NC_00859 rpoB, NT01CX_1107, DNA-directed RNA polymerase subunit
3_226005_226171 beta
pseudomonas_NC_01651 rpoB, EcWSU1_00211, DNA-directed RNA polymerase
4_213592_213738 subunit beta
pseudomonas_NC_00596 rpoB, ACIAD0307, DNA-directed RNA polymerase subunit
6_303883_304054 beta
enterobacter_NC_0146 Entcl_3718, two component transcriptional regulator,
18_3997909_3998085 winged helix family
enterobacter_NZ_GL89
2086_615149_615324
enterobacter_NZ_GL89
2086_1664663_1664834
enterobacter_NZ_GG70
4865_427821_427978
enterobacter_NZ_GL89
2087_1610708_1610874
TABLE 6
Species/strain level probes
Probe Coordinates Binding region 1 Binding region 2
acinetobacter_NC_010611_627997_628164 GCAGCACTTGACCGCCATGAGTGACCA CATCGCACCAACAACAATAATCG
acinetobacter_NC_010611_2417580_2417755 GTGATCACTGATGCACCAGATGAAGT ATCTTGATATTCAAGTCTATGACG
acinetobacter_CP002522_11753_11931 GATATTATTGATCATGGTGCCAAGCCAA CAATATGAAGCTGACGACGCG
acinetobacter_NC_011586_3908329_3908508 GCTGAGCGTGAAGGTTCATGGATTATTA GGTAAGGCTTACGGTCTCAT
acinetobacter_NC_010611_145181_145340 GCATCTTGTGCAGCCTGAATAGCAGCGT ACCACGTTGAATATCACCTTCGG
CAT
acinetobacter_NC_010611_3854494_3854662 AAGTCCATAATTGCTTGAGTGTAGTCAT ATCTTCGCACTGAATAATAAGAA
CAT
acinetobacter_NC_010400_56216_56383 GCTTGCTGGTTCTGCACGTAGCTTACTG AAGATGAACAGGCTACTGCAA
acinetobacter_NC_010611_1454960_1455136 GCAGCGCTGTGCAAGTTCAATGTATTCT CTCGTGCGAGTATTCCTTAAGTGT
acinetobacter_NC_009085_255964_256143 GTATAACACTCGGCCAGCGCCAAGGTTC GTTCACACATCGCCACAATATGAT
clostridium_NC_013974_3097606_3097772 ACCATGCAGATACAATGAACCA GGATGATAAGACACATCCAATTC
clostridium_FN665653_103469_103631 CATCAACAGCTTCTTGAAGCATTCC GTCCAACAACTATAACAGAACGTC
clostridium_NC_013974_117188_117346 AACATATCACCTGATATTCTAGTATC ATTCCATTATATTCAACAGGATT
GTGA
clostridium_NC_013316_3012882_3013047 GCTGTTGCTTGCGGATACTG CGTATATGTAGCTCAAGTTGC
clostridium_FN668375_1212250_1212413 AAGAGCTAATGCAGCTATTGCACTTAT CATACACTTCAGCTATAAGACCAT
clostridium_NC_013315_3754484_3754640 AACAAGAGCAGAAGTTACAGACGT GTATAATGGTGGCTAGAGGTGA
clostridium_FN665654_3239860_3240039 ACTCGTGAAGACCATGCAGATACAA AATACTTACAATGCCTGAGGA
clostridium_FN668941_3228320_3228491 ACCATGCAGATACAATGAACC CCTGAGGATGATAAGACACATC
clostridium_NC_013974_1962664_1962825 GCATCTGCTGCTTCTATTGCTCCTACT ACATGAACTGATATTAGTTCTCC
AA
clostridium_NC_003366_2769687_2769851 GCACAAGCTGGAGATAACATCGG GTAGAGGACGTATTCACAATCACT
clostridium_FN665653_127741_127918 CTCTATCAGCTTCTACTGCTTCTTC CCATCTCATCCACAGTTAATATA
TC
clostridium_NC_013316_2259929_2260107 AGATGAGATTCATACTATCGTTGGAGCT AGCAGAGAGAATAGTAAGAGGAGA
clostridium_NC_009089_94774_94937 CATCAACAGCTTCTTGAAGCATT GTCCAACAACTATAACAGAACG
clostridium_NC_013315_2044225_2044389 GTCAGCAATACGCCACCAAGCTCCTAT GTGGTGGATATCCTGTTACC
clostridium_NC_013315_2299408_2299586 GCGCAATAGAGTTGTATAAGAGTGCTG AGCATTAATTATAGATTATAATG
TATAA
clostridium_FN668941_3244255_3244408 GGCATAATAGGATGGATAGATGA ACTAATCCAACTTCTACTGCTAT
clostridium_NC_013316_3610909_3611065 GTACATTCACATATAGACCATCTTAA ACATAGGTGCAGGTAGAATAGTA
TA
clostridium_FN665653_1104859_1105031 CCATACCAGTATCTTGGCATATTG ATAATGAATAACAGCAGGTGTAT
TA
clostridium_NC_003366_2753681_2753838 AGATGAAGCACAAGCTGGAGATAA AGGACGTATTCACAATCACTG
clostridium_FN665653_710906_711080 ATAATCATTCACCTCCATCATTCATAA ACTGAATATGGTTCGTCTCA
clostridium_NC_009089_3706562_3706720 GTACATTCACATATAGACCATCTTA ACATAGGTGCAGGTAGAATAGT
clostridium_NC_013316_1372812_1372968 ACTCCACCAGGATGTTGTCC GTAGGACCGTCGTGTCCAAG
clostridium_FN665652_676696_676895 GCAATATCAATGGTATCGAAGGCACTAT GTATTGAAGGTACTATTAGCGAT
ATGC
clostridium_NC_013316_2641651_2641808 GTGCCGGTCTCGGTTACTCAATG GGATTATTATAATGCAGCTAGAAG
clostridium_FN668375_3595870_3596026 GTACATTCACATATAGACCATCTT ACATAGGTGCAGGTAGAATAGTA
clostridium_FN668941_1105700_1105868 AGTTCCTTCATATGACTCAGTTGATTGA GTTATATCTTCAATTATACATTC
CTGC
clostridium_NC_013974_2505182_2505359 CAGCAGTTGTTGCTAGAGGTATG GCATCACCAGGTGCAGCAAGT
clostridium_NC_013315_1077126_1077298 GCAATTCTCTGTTGTTGTCCTCCACTCA AGTAAGAGCCTCTTCTTGGTCAT
GA
clostridium_NC_009089_2182303_2182482 CTATTCCTGATAATAAGTGTGTCCTCAT CGGCATCATCTAACAATTCTTCT
clostridium_FN665652_1909777_1909942 GTAATTCCAATTACTTCTAGCTCTGGTG TACCATCTTCTCCATGTGTAT
clostridium_NC_013316_3300896_3301062 CCATGCAGATACAATGAACCAG GATGATAAGACACATCCAATTCC
clostridium_NC_013316_871338_871499 CCTTCTGCCATTGTAGAACAAGCTCCAT CCTGTAACTGTCCACTGAGC
clostridium_NC_013316_3608873_3609047 CAATCATGATAGAATTAGATGGAAC AGCAATAGTTCCATCAGGAGCATC
clostridium_FN665654_3717059_3717221 AGTGGTGAAGGTGTTCAACAAG ACTGAAGCTGGATATGTTGGAG
clostridium_NC_013315_2010489_2010657 CGCCTCTTCAGAAGCGGATATCA GCCAGACTTCCGCCACAACCT
clostridium_NC_013315_3236301_3236474 GGCATAATAGGATGGATAGATGAGC GCAGCAGTTGTACCTACAACTAA
clostridium_NC_013315_1095924_1096090 AGTTCCTTCATATGACTCAGTTGATTG GTTATATCTTCAATTATACATTC
CTGCG
enterobacter_NC_014121_4735453_4735632 GCATGGTAGTTCGCCAGCCGCTGGAAC ACAGCAACCGCAAGTTCTTGACAT
enterobacter_NC_015663_1014187_1014345 AATATCATGGTCGTGTCCAGGCACTGGC GTTCTGGTAGCTGCTTCTACTGTA
enterobacter_FP929040_3448334_3448513 AACTTACAACTACGCGCACTTGAATCG GAGTGTTGTATGATAGTCTCGGT
enterobacter_NC_009436_4051820_4051985 GCAAGTTGAGGAGATGCTGGCATGATTC ACATGGCTCTGGAAGATGTGCTG
ATC
enterococcus_FP929058_1738439_1738606 GCGATAATTGTAATGATTCGTGGTGTTA CCGTTGTCAATCCAGTTAGTAGA
CT
enterococcus_CP002621_1819224_1819388 ACTGTGGCAGTCTATGTTCCAATTGTA CTTATCGACATAATCCTGATAATC
enterococcus_FP929058_904007_904173 GCGTCGCTTCTTGCGCTCGCC AATGTATTCATACCGTCAAGT
enterococcus_FP929058_551757_551920 GCCTTCACAACTACGTTGGAAGGTCTTC CTAACAGTCCTGCCGACTAC
enterococcus_NC_004668_1122345_1122507 GCCTTCACAACTACGTTGGAAGGTCTT CTAACAGTCCTGCCGACTACT
klebsiella_NC_009648_2885456_2885620 GCCGCTGAGCGGCGGCAAGCCGATGGC GAATGGCAGGCCAAGCTGAAGGCG
klebsiella_NC_009648_3899012_3899182 GCCAAGCGGCATTCTGGCGCCAGTGGA CCAGACCGGAGTGGACAACGTCG
AGGCG
klebsiella_NC_009648_4980596_4980757 GCCGTATATCATCGGCAATAACCGCACG GCATGATGGTCAACAAGGTGC
klebsiella_NC_009648_3266359_3266519 ACGAGCCGAGATAGGTCTGCAGCGTAC GTACTGATATTCACCATACTGCCG
klebsiella_NC_012731_2557467_2557634 GCAATATCTTCACCGGCAGCCACCGCG GGTATATGGCACGCCAATCGC
klebsiella_NC_012731_4857136_4857315 AATAACCTTAACGTCGCCAACACG CTCGGTGAACACCTCCTGGCACG
proteus_NC_010554_547938_548117 GCGGAACTGCTTGGCGTAGTAAGC CATGTAGTGCCGTAGACCTTCAC
CA
pseudomonas_NC_008463_658500_658676 GCGAGACCGGCGGCACCATCGTCTCCAG TTCTGCCTGATGGACGTCTCCGG
CTCG
pseudomonas_NC_008463_753931_754099 GCGGTTCACCTGTTCGCCTTCGAACACG GCGCAGCATCTGACGCAGGATGG
TCTCG
pseudomonas_NC_009656_6431649_6431828 ACTCCATCGCCATCAAGGACATGGCCGG ATCGACGTGTTCCGCATCTTCGA
CGCG
pseudomonas_NC_008463_560357_560534 GCCTGATGCACTACAGCGCCTGG TACCACATGGTCGATCTCGACGA
CTGC
pseudomonas_NC_010322_5224859_5225023 GCGCATCCAGGACGGCGAGTACG CTTCGAGTGCCTGCACGAGCTGAA
pseudomonas_NC_008463_4839746_4839924 GCTGGAGAACGTCAAGGTGGTGATCATC ACCGATAACGACGACCGCATCAA
staph_FN433596_2844085_2844263 ACGATTGGAGAAGGCAGTGTGATTGG GGACAGATTACAATTGGCG
staph_NC_009632_1198350_1198529 GCCGCAATACCGATATTCCA CCATTGTCCACCAGCTGAACCG
staph_FN433596_2521244_2521419 GTGAAGGTCGTGCTCCTATCGGT AGATCTGGTGAAGTTCGTATGAT
staph_NC_009487_430842_431017 GCTGGTACTTGTACTTATATCGA ATCAGAAGATGATATCGTTACGT
CAT
staph_NC_009782_2086681_2086849 GCGCATATTGCATTAATGGCTATAGAT GCCAGCAGGTTATACACTCG
staph_NC_009782_58256_58423 GCAATTCTTACCACAGCACGAAGAACAG ATCTAGATGAAGATAATGAAGTCG
staph_NC_013450_991049_991222 GCATCTTCATACAATACTTCTAGCTTAC CACAATACCAGTTGTATTACG
staph_NC_013450_1360842_1361008 GCTTCAGCGCCATTACCGCCACCAGCT ACTCTTGATATATTCTTGTAAGCG
staph_AM990992_2526026_2526192 GTTCACACAACGCGCCGACTAGAATCC CACGATATCCAAGATAATGATTG
GCTA
staph_NC_010079_361284_361447 GCGCACCTACAATCGCCATTACTACAC ACTCATTATCGACTGTTACATCG
ACTGA
staph_NC 007795_2085723_2085901 AGCGCACATGTGACAGCGTGTAGGTTA GTGCCTTAGATTGTTCAGAACAAT
staph_NC_009641_23125_23297 CGAATGGATATGTACCATGGTCGATATC CTCTCTAATATGATGTCCAT
staph_FN433596_2144570_2144734 ACTACAACAGCAACCGCATTACAATGGC GGTGCTAAGAGGTCATCGGA
staph_NC_009782_54857_55020 AGCTTCAGATAAGTACCTATCTGA GGAAGAATAGTTATTCTTGATAA
TGTAT
staph_AM990992_1656616_1656789 CGTATTGCTCGAATACATGATA ACAATGTATCAAGGCCAGCT
staph_NC_007793_44227_44395 GCGACCAGTTGTTATCGACCGTGT CAGAACGATACGGTGCTGTATA
staph_NC_009641_1102949_1103116 CAATTACATTGTCTGTTGCGTAGATACC GTTGTGGCTAATGTGCCAGTT
staph_NC_009641_1137731_1137898 GCACCACTCTATAGCAGTAGCGTATTG ACAGCCAATGTCACCTAAGTCAA
CA
staph_FN433596_2715713_2715871 ACAGTCCGAATAAGATACGACTATTCGA CGTTGTAACGTATATGAATAGTT
GA
staph_NC_009782_606652_606825 AGATGCAATAACAGGTCGAATATTAATT GCCATAGTGAGAGTAGTGAA
staph_FN433596_657625_657803 AGATGCAATAACAGGTCGAATATTAA ACACATACGGCCATAGTGAGAG
pseudomonas_NC_008463_4756080_4756240 GAATCGAACGGTCTCATTAACAGAT GCTTTCCAGGGATATAAGACGC
pseudomonas_NC_002516_1063894_1064077 CCCGCAGAGTCACACTCGGA ACTCTTGGTACTACTCACTAGC
pseudomonas_NC_008463_3182693_3182865 GAGTCTCTTTCAACCTGGATTAGATAT AAGATTAATAGCGTACTTTACTCC
pseudomonas_NC_009656_2819490_2819655 ATCCCGCAGATACTAGGTTCTTAAT GAACTATTCATATTACACCCTAA
GG
pseudomonas_NC_008463_3184022_3184185 CAGTGGGCTATCCTAAGCCAAAG CATAAGCGAACTAACTATCACTTA
pseudomonas_NC_002516_1065937_1066093 ACAAAGCGTTCTAAACGATTAGAACT CGAGAAAGGAAACAGGATAGTAC
pseudomonas_NC_002516_1067833_1068007 CCAATGGAGAAGTCTAAATGTCCAA TTATCAGAGATACATGACTCTTA
GG
pseudomonas_NC_008463_3182351_3182508 CGAATCACTGGACTACATTTATATTTCT AGCGAACCTTTATATTTGACCAT
pseudomonas_NC_008463_3184314_3184473 CTCAAGTCTTGCCCTGATAGAATTAT TCACGACTTATCTACTTTAGAAA
TC
pseudomonas_AP012280_3765216_3765383 GGTGATCGTTATTATGATAGTACGGC CTCGGTTAAGGGAATTACGAC
pseudomonas_AP012280_3765033_3765192 ACTCGGATGGTAGGTTTATTAAAGC GTGATCGTTATTATGATAGTACGG
enterococcus_NZ_GG703715_13422_13573 ACAATCGTTGTCGCACTGCATAG GAACTTGGTCTACCGTACCAC
enterococcus_NZ_GG703582_76982_77140 GGATAATACAATCCTAATACGTACGGA GCTGCTGTAACTAGGGTAGC
enterococcus_NZ_GL455004_28219_28381 CTATATTCAACGGGTCACGGGTAG TCATTGATTCGATCTCGTAACTC
enterococcus_NZ_GG703720_94699_94852 AATGTTATTGTGGTTGCGTGTTCG TACTTTGGAAGTGCCCTGAC
enterococcus_NZ_GG703715_15795_15951 CATGTCTTCTAGTACAGGTTTGCCG TGTAAGAGGCCGCTAACTTC
enterococcus_NZ_GL455899_32848_32984 CTCTGGCTCGTGGGCTCGG TTCTTGAGATAGTCCGGTATAATC
enterococcus_NZ_GG692918_325104_325257 ATTCGATCACGATGGGCTGGG AATTTCCTGTGTCATACACGC
enterococcus_NC_004668_920608_920750 CAATTGATTTAGCCACTACACCTTAC CACTATTCTGGCGACCACC
enterococcus_NZ_GG703575_78829_78963 GATAAAGAAGCGTCTTGACCCAGT ATCTGGTGCTCCTTGACGC
enterococcus NZ_GL455931_26355_26493 GCAAATTTAGAGAGTGCATGCATG GGAAGAGGACGGCATACAAC
enterococcus_NZ_GG669058_207026_207172 CATTTCATCTAGACCGCTCGTGT GCTTGAAGTGTATGTTGGGAC
proteus_NZ_GG661998_111187_111342 GTCGCCCTCGTGCTAACGT GGTTCTTTGATGTACCGGTT
proteus_NC_010554_2037943_2038091 GCTGATGACGGTGAAGTTTATCA CATTATCGCACATATTGACCAC
proteus_NZ_GG668576_810893_811054 GAAATTAGCTAAAGGGATATCGCG AACTTTCCGCCAATCCTGC
proteus NZ_GG668594_760_939 CACCTACGTTCTCACCTGCAC ATTCGATAGTACCAGTTACGTC
proteus_NZ_GG668579_22072_22234 GTTGCTTATAGCGTCGCTGCT CTGGTTATCGAGAAGATAAAGG
proteus NC_010554_2448957_2449119 GTAAGCGTAGCGATACGTTGAG GAGTGAACGCACCACTGG
proteus_NC_010554_3033758_3033936 TCAGGTAGAGAATACTCAGGCGC CGGAGAAGGCTAGGTTGTC
proteus_NC_010554_454391_454540 GCAACCCACTCCCATGGTGT CGTTCTTCATCAGACAATCTG
pseudomonas_NC_009085_307050_307218 AACTAAACCTACACGGAATTGGTTC GCAGATACACGACGTTTATGT
pseudomonas_NC_009085_308225_308377 GCCGCTTCACCTACGTTAGGAA CGTAAAGATGAGTCTTTAACGTC
pseudomonas_NC_016612_1674334_1674490 GACGTTTGTGCGTAATCTCAGAC GAGGAAACCGTATTCGTTCGT
pseudomonas_NC_016603_3425179_3425337 ACAACACTTTACCACTTGAGTGGG GTAACTGCCCATGTCAAGATAC
pseudomonas_NC_016603_3427629_3427808 CCACGTTTAGTTGAACCACCGC TCAATACGCCAGTTGTTAGTTC
pseudomonas_NC_010410_3543925_3544088 AATCGATAATAAGTACGGTGCATCC GAAGAATACATTCGCGTACATC
pseudomonas_NC_005966_304936_305079 AAGCAAGATCGAGTCTTCATAGTTG GATATACACGATACCTGATTCGT
pseudomonas_NC_008593_226005_226171 CCGATATTCATACGAGAAGGTACAC CAGTAACTCTATTGTCAAACGGT
pseudomonas_NC_016514_213592_213738 GTAGTGAGTCGGGTGTACGTCTC TCTTCGATAGCAGACAGATAGT
pseudomonas_NC_005966_303883_304054 ACCTACACGGAATTGGTTCTCAGT GATACACGACGTTTGTGTGTA
enterobacter_NC_014618_3997909_3998085 CAACATCATTAGCTTGGTCGTGGG TTGCGTGTTACCAACTCGTC
enterobacter_NZ_GL892086_615149_615324 CGGCACGTCCGAATCGTATCA TCGTGTCCCGTATATGTTGG
enterobacter_NZ_GL892086_1664663_1664834 AATAGAGGCCCACAAGTCTTGTTC CGCTCTCCACTATGGGTAGT
enterobacter_NZ_GG704865_427821_427978 GCTACATTAATCACTATGGACAGACA GATGGTCGATCTATCGTCTCT
enterobacter_NZ_GL892087_1610708_1610874 GAAGTGTTATTCAAACTTTGGTCCC CTTGAACCCTTGGTTCAAGGT
TABLE 7
Marker regions are highly polymorphic regions (like, e.g., VNTRs) that provide
fine resolution.
Probe Coordinates Gene
plasmids_NC_011980_58308_58487 insA, MM1_0111, IS1 protein InsA, MM1_0112, IS1
protein InsB
plasmids_NC_015599_37281_37455 intIl, pN3_046, integrase
plasmids_NC_007351_37979_38146 SSPP128, IS431 transposase
plasmids_FN822749_1846_2009 ETEC1392/75_p75_00003, putative IS100
transposase
plasmids_NC_004851_143949_144109 CP0039, IS629 ORF2
plasmids_NC_010558_156799_156957 IS1-insB, IPF_205, IS1-insB
plasmids_NC_012547_53585_53752 tnpA, PGO1_p15, putative transposase TnpA
plasmids_NC_013950_91008_91174 tnpR, pKF94_116, TnpR
plasmids_NC_002698_168967_169123 insB, pWR501_0054, IS1 transposase
CMY_AB061794_343_489 intI1, DNA integrase
IMG_AY033653_1343_1500 intI1, DNA integrase
TEM_U36911_4374_4551
TEM_U36911_7596_7762
TABLE 8
Marker probes
Probe Coordinates Binding region 1 Binding region 2
plasmids_NC_011980_58308_58487 GCAGTCGGTAACCTCGCGC GCGCTATCTCTGCTCTCACTGC
plasmids_NC_015599_37281_37455 GCTGTAATGCAAGTAGCGTATGCGCTC GAACAGCAAGGCCGCCAATGCCTGACG
plasmids_NC_007351_37979_38146 CGCATATGCTGAATGATTATCTCGTTGC ATCTTGCTCAATGAGGTTATTCA
plasmids_FN822749_1846_2009 GACGACAGATGCAGGTTGA CGCATCGCCGATGCTCATC
plasmids_NC_004851_143949_144109 CGCCTGCTCCAGTGCATCCAGCACGAAT ATGCTCTCCGCCATCGCGTTGTCA
plasmids_NC_010558_156799_156957 AGTGCGTTCACCGAATACGTGCGCA CAGGTTATGCCGCTCAATTC
plasmids_NC_012547_53585_53752 CGCATATGCTGAATGATTATCTCGTTG ACGGTGATCTTGCTCAATGAGGTTATTC
plasmids_NC_013950_91008_91174 GCTGTGGCACAGGCTGAACGCCG GGTGATGTCATTCTGGTTAAGA
plasmids_NC_002698_168967_169123 ACATAATCTGAATCTGAGACAACATC ACGCACTCTGGCCACACTGG
CMY_AB061794_343_489 CATCACGAAGCCCGCCACA GCCCTTGAGCGGAAGTATC
IMG_AY033653_1343_1500 CGGAAGTATCCGCGCGCC TTCGATCACGGCACGATC
TEM_U36911_4374_4551 CATTCTCTCGCTTTAATTTATTAACCT ATCGACCTTCTGGACATTATC
TEM_U36911_7596_7762 CGTTGCTTACGCAACCAAATATC TGATCTTGCTCAATGAGGTTA
TABLE 9
Resistance regions can be used to detect one or more genes associated with
resistance to antimicrobial compounds, such as antibiotic resistance genes.
Probe Coordinates Gene
plasmids_NC_013950_90185_90338 pKF94_115, beta-lactamase
plasmids_NC_013452_4052_4209 SAAV_b4 tetracycline resistance protein
plasmids_NC_014208_52313_52469 pKOX105p23, VIM-1, pKOX105p24, IntIA
pKOX105p67, truncated AadA
betalactamase_AB372224_738_905 blaCMY-39, class C beta-lactamase CMY-39
betalactamase_EF685371_398_548 beta-lactamase CMY-29
betalactamase_DQ149247_231_371 bla-OXA-86, OXA-86
betalactamase_AY750911_244_414 bla-oxa-69, beta-lactamase OXA-69
betalactamase_DQ519087_417_575 blaOXA-93, beta-lactamase OXA-93
betalactamase_AM231719_379_537 blaOXA-90, class D beta lactamase
betalactamase_Y14156_663_819 CTX-M-4, beta lactamase
betalactamase_JN227085_763_931 blaCTX-M-117, CTX-M-117 beta-lactamase
betalactamase_EU259884_1030_11 aacA4, AacA4 aminoglycoside (6')
70 acetyltransferase
betalactamase_HQ913565_578_730 blaCTX-M-106, beta-lactamase CTX-M-106
betalactamase_AY524988_385_552 blaVIM-9, VIM-9
CARB_AF030945_646_795 CARB-6, class A beta-lactamase
CARB_U14749_1227_1390 blaCARB-4, CARB-4 precursor
CARB_AF313471_2731_2906 aadA1a, AAD(3″) aminoglycoside (3″)
adenylyltransferase
CMY_DQ463751_613_790 blaCMY-23, hypothetical CMY-23 protein
precursor
CMY_EF685371_397_552 beta-lactamase CMY-29
CMY_EU515251_583_733 blaCMY-40, AmpC beta-lactamase
CMY_JN714478_1882_2055 blaCMY-66, AmpC beta-lactamase CMY-66
CMY_X91840_1872_2046 bla CMY-2, extended spectrum beta-lactamase
CTXM_EF219134_13713_13858 AadA2 aminoglycoside adenylytransferase;
confers resistance to streptomycin and
spectinomycin
CTXM_HQ398215_802_947 blaCTX-M-98, beta-lactamase CTX-M-102
CTXM_AM982522_639_788 blaCTX-M-78, CTX-M-78 beta-lactamase
GES_HM173356_1163_1321 blaGES-16, carbapenem-hydrolyzing extended-
spectrum beta lactamase GES-16
GES_AF156486_1754 1905 ges-1, beta-lactamase GES-1
GES_HQ874631_571_748 extended-spectrum beta-lactamase GES-17
GES_FJ820124_1174_1338 beta-lactamase GES10
IMG_DQ361087_489_645 blaIMP-22, metallo-beta-lactamase IMP-22
IMG_JN848782_301_475 blaIMP-33, metallo-beta-lactamase IMP-33
IMG_EF192154_182_328 blaIMP-24, metallo-beta-lactamase IMP-24
IMG_AF318077_871_1047 aacC4, aminoglycoside-N-
acetyltransferase
IMG_AF318077_515_657 aacC4, aminoglycoside 6'-N-
acetyltransferase
KPC_HM066995_226_375 b1aKPC, beta-lactamase KPC-11
KPC_GQ140348_624_799 KPC-10, beta-lactamase KPC-10
KPC_EU729727_683_840 carbapenem-hydrolyzing beta-lactamase KPC-
7
KPC_FJ234412_691_839 blaKPC-8, beta-lactamase KPC-8
NDM_JN104597_64_211 blaNDM-5, NDM-5 metallo-beta-lactamase
NDM_FN396876_2744_2885 blaNDM-1, metallo-beta-lactamase
NDM_FN396876_2958_3117 blaNDM-1, metallo-beta-lactamase
NDM_JN104597_314_465 blaNDM-5, NDM-5 metallo-beta-lactamase
NDM_FN396876_2382_2548 blaNDM-1, metallo-beta-lactamase
OXA_EF650035_239_388 bla-OXA-109, beta-lactamase OXA-109
OXA_EU019535_389_537 bla-OXA-80, beta-lactamase OXA-80
OXA_EF650035_423_594 bla-OXA-109, beta-lactamase OXA-109
OXA_DQ309276_232_380 bla-OXA-84, beta-lactamase OXA-84
OXA_DQ445683_232_380 bla-OXA-89, oxacillinase OXA-89
OXA_X75562_201_366 OXA-7, beta lactamase OXA-7
OXA_M55547_995_1154 tnpR, aac, Aac
OXA_AY445080_313_469 blaOXA-56, restricted-spectrum beta-
lactamase OXA-56
PER_Z21957_217_371 PER-1, extended-spectrum beta-lactamase
PER-1
PER_HQ713678_6002_6167 blaPER-7, blaPER-7
PER_GQ396303_667_844 blaPER-6, extended-spectrum beta-lactamase
PER-6
PER_X93314_954_1122 bla(per-2), extended-spectrum beta-
lactamase
PER_HQ713678_4517_4674 transposase
PER_HQ713678_5074_5219 transposase
PER_GQ396303_254_399 blaPER-6, extended-spectrum beta-lactamase
PER-6
SHV_AY661885_656_806 blaSHV-30, beta-lactamase SHV-30
SHV_AF535128_587_761 blaSHV-40, beta-lactamase SHV-40
SHV_U92041_406_579 SHV-8, beta-lactamase
SHV_AY288915_617_764 blaSHV-50, beta-lactamase SHV-50
SHV_HQ637576_88_245 blaSHV-135, beta-lactamase SHV-135
SHV_AF535128_188_362 blaSHV-40, beta-lactamase SHV-40
SHV_X98102_763_913 blaSHV-2a, beta-lactamase SHV-2a
TEM_GQ149347_3605_3747 near kanamycin resistance protein
TEM_GU371926_11801_11944 traN, TraN
TEM_J01749_766_908 tet, tetracycline resistance protein
VEB_EU259884_6947_7094 blaVEB-6, VEB-6 extended-spectrum beta-
lactamase
VEB_EF136375_596_738 blaVEB-4, extended-spectrum beta-lactamase
VEB-4
VEB_EF420108_234_380 blaVEB-5, extended spectrum beta-lactamase
VEB-5
VEB_AF010416_89_230 veb-1, extended spectrum beta-lactamase
VIM_AY524988_385_552 blaVIM-9, VIM-9
VIM_Y18050_3464_3614 blaVIM, beta-lactamase VIM-1
VIM_AY635904_58_203 blaVIM-11, metallo-beta-lactamase
VIM_HM750249_275_454 bla, metallo-beta-lactamase VIM-25
VIM_AJ536835_313_481 blaVIM-7, metallo-b-lactamase
VIM_EU118148_131_300 near intI1, DNA integrase INTI1
VIM_DQ143913_921_1063 near intI1, IntI1
VIM_EU118148_1060_1229 blaVIM-17, metallo-beta-lactamase VIM-17
van_NC_008821.1_11898_12045 vanB, pVEF236, D-alanine--D-lactate ligase
mecA_AY820253.1_1431_1608 mecA, PBP2a-like protein
mecA_AY952298.1_130_302 Pbp2′
erm_NC_002745.2_871803_871973
erm_NC_002745.2_871666_871841 ermA, SA1951, rRNA methylase Erm(A)
TABLE 10
Resistance probes
Probe Coordinates Binding region 1 Binding region 2
plasmids_NC_013950_90185_90338 GAGGACCGAAGGAGCTAACCG CGCCGCATACACTATTCTC
plasmids_NC_013452_4052_4209 CTCATTCCAGAAGCAACTTCTTCTT GGATAGCCATGGCTACAAGAATA
plasmids_NC_014208_52313_52469 GGTTCTGGACCAGTTGCGTGAGCGC CGTAACATCGTTGCTGCTCCAT
betalactamase_AB372224_738_905 CGCTGGATTTCACGCCATAGGC TGTCGCTACCGTTGATGATT
betalactamase_EF685371_398_548 CGTATAGGTGGCTAAGTGCAGC GTAACTCATTCCTGAGGGTTTC
betalactamase_DQ149247_231_371 GTACATACTCGATCGAAGCACGA CCGGAATAGCGGAAGCTTTC
betalactamase_AY750911_244_414 AAGGTCGAAGCAGGTACATACTCG AGACATGAGCTCAAGTCCAAT
betalactamase_DQ519087_417_575 GAAGCTTTCATAGCGTCGCCTAG TTAGCTAGCTTGTAAGCAAATTG
betalactamase_AM231719_379_537 GAAGCTTTCATGGCATCGCCTAG AGCTAGCTTGTAAGCAAACTG
betalactamase_Y14156_663_819 CGCTACCGGTAGTATTGCCCTT AGAATATCCCGACGGCTTTC
betalactamase_JN227085_763_931 ATCGCCACGTTATCGCTGTACT TTTACCCAGCGTCAGATTCC
betalactamase_EU259884_1030_1170 CAAGTACTGTTCCTGTACGTCAGC TCGCCAGTAACTGGTCTATTC
betalactamase_HQ913565_578_730 CAACGTCTGCGCCATCGCC CGCAATATCATTGGTGGTGC
betalactamase_AY524988_385_552 GCCGCCCGAAGGACATCAAC CAGACGGGACGTACACAAC
CARB_AF030945_646_795 CGTGCTGGCTATTGCCTTAGG GTAATACTCCTAGCACCAAATC
CARB_U14749_1227_1390 CATTAGGAGTTGTCGTATCCCTCA AATACTCCGAGCACCAAATC
CARB_AF313471_2731_2906 AAATTGCAGTTCGCGCTTAGC GTTCCATAGCGTTAAGGTTTC
CMY_DQ463751_613_790 GCGCCAAACAGACCAATGCT GATTTCACGCCATAGGCTC
CMY_EF685371_397_552 GTATAGGTGGCTAAGTGCAGCA TCGTAACTCATTCCTGAGGG
CMY_EU515251_583_733 GTCATCGCCTCTTCGTAGCTC GCCATATCGATAACGCTGG
CMY_JN714478_1882_2055 ACCAATACGCCAGTAGCGAGA GCAACGTAGCTGCCAAATC
CMY_X91840_1872_2046 CAATCAGTGTGTTTGATTTGCACC TACCCGGAATAGCCTGCTC
CTXM_EF219134_13713_13858 CGGATAACGCCACGGGATGA ACCGGGTCAAAGAATTCCTC
CTXM_HQ398215_802_947 GCGGCGTGGTGGTGTCTC CGCTGCCGGTCTTATCAC
CTXM_AM982522_639_788 GCCACGTCACCAGCTGCG CGGCTGGGTGAAGTAAGTC
GES_HM173356_1163_1321 GCTCGTAGCGTCGCGTCTC TTGACCGACAGAGGCAAC
GES_AF156486_1754_1905 CAGCAGGTCCGCCAATTTCTC AGTGGACGTCAGTGCGC
GES_HQ874631_571_748 CCATAGAGGACTTTAGCCACAGT TACACCGCTACAGCGTAAT
GES_FJ820124_1174_1338 CATATGCAGAGTGAGCGGTCC TCAATTCTTTCAAAGACCAGC
IMG_DQ361087_489_645 CCATTAACTTCTTCAAACGATGTATG ACCCGTGCTGTCGCTAT
IMG_JN848782_301_475 GTGCTGTCGCTATGGAAATGTG AACCAAACCACTAGGTTATCTT
IMG_EF192154_182_328 GTCAGTGTTTACAAGAACCACCA ATGCATACGTGGGAATAGATT
IMG_AF318077_871_1047 CGAACCAGCTTGGTTCCCAAG TCACTGCGTGTTCGCTC
IMG_AF318077_515_657 GATGCTGTACTTTGTGATGCCTA CGCTTGGCAAGTACTGTTC
KPC_HM066995_226_375 GCAAGAAAGCCCTTGAATGAGC GCGTTATCACTGTATTGCAC
KPC_GQ140348_624_799 AATCAACAAACTGCTGCCGCT GCTGTACTTGTCATCCTTGT
KPC_EU729727_683_840 CCAGTCTGCCGGCACCGC TCGAGCGCGAGTCTAGC
KPC_FJ234412_691_839 CCGACTGCCCAGTCTGCCG CGAGCGCGAGTCTAGCC
NDM_JN104597_64_211 GTAAATAGATGATCTTAATTTGGTTCAC TTGCTGGCCAATCGTCG
NDM_FN396876_2744_2885 CACAGCCTGACTTTCGCCGC CAAGCAGGAGATCAACCTGC
NDM_FN396876_2958_3117 GGTGGTCGATACCGCCTGG GTGAAATCCGCCCGACG
NDM_JN104597_314_465 CATGTCGAGATAGGAAGTGTGC TGATGCGCGTGAGTCAC
NDM_FN396876_2382_2548 CAATCTGCCATCGCGCGATT CGGCAATCTCGGTGATGC
OXA_EF650035_239_388 CGAAGCAGGTACATACTCGGTC ACGAGCTAAATCTTGATAAACTT
OXA_EU019535_389_537 TAGAATAGCGGAAGCTTTCATGG AGCTAGCTTGTAAGCAAACTG
OXA_EF650035_423_594 CAAGTCCAATACGACGAGCTAAA GAATAGCATGGATTGCACTTC
OXA_DQ309276_232_380 GGTACATACTCGGTCGAAGCAC AATCTTGATAAACTGAAATAGCG
OXA_DQ445683_232_380 GGTACATACTCGGTCGATGCAC TCTTGATAAACCGGAATAGCG
OXA_X75562_201_366 GTAATTGAACTAGCTAATGCCGTAC TTATGACACCAGTTTCTAGGC
OXA_M55547_995_1154 CAAGTACTGTTCCTGTACGTCAG GCCCAGTTGTGATGCATTC
OXA_AY445080_313_469 TCTCTTTCCCATTGTTTCATGGC TGCGGAAATTCTAAGCTGAC
PER_Z21957_217_371 GTAGGTTATGCAGTTATTAGGTTCAG GACTCAGCCGAGTCAAGC
PER_HQ713678_6002_6167 GCAGTACCAACATAGCTAAATGC AAATAACAAATCACAGGCCAC
PER_GQ396303_667_844 GGTCCTGTGGTGGTTTCCACC CGCGATAATGGCTTCATTGG
PER_X93314_954_1122 TAACCGCTGTGGTCCTGTGG TGCGCAATAATAGCTTCATTG
PER_HQ713678_4517_4674 GGAAGCGTTGCTTGCCATAGT AACCGAAGCACCATGTAATT
PER_HQ713678_5074_5219 GTTCGGTGCAAAGACGCCG TCGCAGACTTCAATATCAATATT
PER_GQ396303_254_399 CACCTGATGCAGAACCAGCAT AGGCCACGTTATCACTGTG
SHV_AY661885_656_806 CAGCTGCCGTTGCGAACG CGCAGATAAATCACCACAATC
SHV_AF535128_587_761 GCTCAGACGCTGGCTGGTC CCGCAGATAAATCACCACG
SHV_U92041_406_579 GCCAGTAGCAGATTGGCGGC GAACGGGCGCTCAGACG
SHV_AY288915_617_764 CCACTGCAGCAGATGCCGT GTATCCCGCAGATAAATCACC
SHV_HQ637576_88_245 TTAATTTGCTTAAGCGGCTGCG CCAGCTGTTCGTCACCG
SHV_AF535128_188_362 GGGAAAGCGTTCATCGGCG TCGCTCATGGTAATGGCG
SHV_X98102_763_913 TCTTATCGGCGATAAACCAGCC CGTTGCCAGTGCTCGAT
TEM_GQ149347_3605_3747 GTCGGAAAGTTGACCAGACATTA ATACTAGGAGAAGTTAATAAATACG
TEM_GU371926_11801_11944 GTGAAGTGAATGGTCAGTATGTTG AGTGCGCAGGAGATTAGC
TEM_J01749_766_908 CCTGTCCTACGAGTTGCATGAT ATAATGGCCTGCTTCTCGC
VEB_EU259884_6947_7094 CAAATACTAAATTATACAGTATCAGAG ATGCAAAGCGTTATGAAATTTC
AG
VEB_EF136375_596_738 GTTCTTATTATTATAAGTATCTATTAA CATTAGTGGCTGCTGCAAT
CAGTT
VEB_EF420108_234_380 CATCGGGAAATGGAAGTCGTTAT GTTCAATCGTCAAAGTTGTTC
VEB_AF010416_89_230 CGTGGTTTGTGCTGAGCAAAG CAAAGTTAAGTTGTCAGTTTGAG
VIM_AY524988_385_552 GCCGCCCGAAGGACATCAA AGACGGGACGTACACAAC
VIM_Y18050_3464_3614 GCAACTCATCACCATCACGGA TGATGCGTACGTTGCCAC
VIM_AY635904_58_203 GCGACAGCCATGACAGACGC GGACAATGAGACCATTGGAC
VIM_HM750249_275_454 AAACGACTGCGTTGCGATATG TTCCGAAGGACATCAACGC
VIM_AJ536835_313_481 ATGCGACCAAACGCCATCGC ATCGTCATGGAAGTGCGTA
VIM_EU118148_131_300 GAACAGGCTTATGTCAACTGGG CATAACATCAAACATCGACCC
VIM_DQ143913_921_1063 ACGAACCGAACAGGCTTATGTC TAACGCGCTTGCTGCTT
VIM_EU118148_1060_1229 CATCATAGACGCGGTCAAATAGA ACTCATCACCATCACGGAC
van_NC_008821.1_11898_12045 CAGGCTGTTTCGGGCTGTGA GGGTTATTAATAAAGATGATAGGC
mecA_AY820253.1_1431_1608 TAATTCAAGTGCAACTCTCGCAA TTTATTCTCTAATGCGCTATATATT
mecA_AY952298.1_130_302 GGATAGTTACGACTTTCTGCTTCA TGTATTGCTATTATCGTCAACG
erm_NC_002745.2_871803_871973 GTCAGGCTAAATATAGCTATCTTATCG TCAGTTACTGCTATAGAAATTGAT
erm_NC_002745.2_871666_871841 CATCCTAAGCCAAGTGTAGACTC AAGATATATGGTAATATTCCTTATA
AC
TABLE 11
Additional regions may be used for additional discrimination and
characterization of organisms.
Probe Coordinates Gene
peGFP_N1_730_925
CMY_X92508_126_301
TEM_X64523_2037_21 near tnpR, resolvase
91
TEM_J01749_2068_22 near ROP protein
39
TEM_AF091113_1529_
1699
TEM_J01749_1634_17
83
TEM_U36911_6901_70
69
TEM_GU371926_33909_ klcA, KlcA
34082
VIM_EU118148_2821_ qacEdeltal, quarternary ammomium compound-resistance
2961 protein QacEdeltal sull, dihydropteroate synthase SUL1
van_DQ018710.1_648
1_6652
van_DQ018710.1_676
4_6926
van_AY926880.1_364
0_3785
van_FJ545640.1_517_
690
van_AE017171.1_347
15_34859
van_FJ349556.1_560
1_5765
mecA_AM048806.2_15
74_1720
mecA_EF692630.1_23
9_405
mex_AF092566.1_371_
520
mex_AF092566.1_50_
193
mex_CP000438.1_487
178_487357
mex_NZ_AAQW0100000
1.1_461304_461466
erm_EU047809.1_79_
229
gyrB_NC_015663_145 EAE_24795, hemagluttinin domain-containing
5472_1455621 protein, gyrB, EAE_07020, DNA gyrase subunit B
gyrB_NC_010410_421 gyrB, ABAYE0004, DNA gyrase, subunit B
5_4366
gyrB_NC_005773_490 gyrB, PSPPH_0004, DNA gyrase subunit B
4_5052
gyrB_NC_016514_534 gyrB, EcWSU1_00004, DNA gyrase subunit B
3_5487
gyrB_NC_016603_263 gyrB, BDGL_002434, DNA gyrase, subunit B
1439_2631616
gyrB_NC_009436_436 gyrB, Ent638_0004, DNA gyrase subunit B
6_4524
gyrB_NC_009512_420 gyrB, Pput_0004, DNA gyrase subunit B
3_4373
TABLE 12
Additional arms
Probe Coordinates Binding region 1 Binding region 2
peGFP_N1_730_925 GTGGTATGGCTGATTATGATCTAGAGT GAGTTTGGACAAACCACAACTAGAA
CMY_X92508_126_301 AGTATCTTACCTGAAATTCCCTCAC CCTCTCGTCATAAGTCGAATG
TEM_X64523_2037_2191 CAGTCCCTCGATATTCAGATCAGA TTAACAATTTCGCAACCGTC
TEM_J01749_2068_2239 CAGCTGCGGTAAAGCTCATCA CATAGTTAAGCCAGTATACACTC
TEM_AF091113_1529_1699 GTAACAACTTTCATGCTCTCCTAAA CGGTAACTGATGCCGTATTT
TEM_J01749_1634_1783 CGTTTCCAGACTTTACGAAACAC ACGTTGTGAGGGTAAACAAC
TEM_U36911_6901_7069 CATCATGTTCATATTTATCAGAGCTC TAGATTTCATAAAGTCTAACACAC
TEM_GU371926_33909_34082 GTTTCCACATGGTGAACGGTG AAACCTGTCACTCTGAATGTT
VIM_EU118148_2821_2961 GCTGTAATTATGACGACGCCG CTCGGTGAGATTCAGAATGC
van_DQ018710.1_6481_6652 GTGTATGTCAGCGATTTGTCCAT TGTCATATTGTCTTGCCGATT
van_DQ018710.1_6764_6926 GTCCACCTCGCCAACAATCAA ATATCAACACGGGAAAGACCT
van_AY926880.1_3640_3785 GCGTGATTATCACGTTCGGCA CTTGCAGATTTAACCGACAC
van_FJ545640.1_517_690 GGCTCGACTTCCTGATGAATACG TGAAACCGGGCAGAGTATT
van_AE017171.1_34715_34859 CAACGATGTATGTCAACGATTTGT ATTGCGTAGTCCAATTCGTC
van_FJ349556.1_5601_5765 GGCTCGGCTTCCTGATGAATAC AGGCATGGTATTGACTTCATT
mecA_AM048806.2_1574_1720 CAGTATTTCACCTTGTCCGTAACC GTTTACGACTTGTTGCATGC
mecA_EF692630.1_239_405 AATGTTTATATCTTTAACGCCTAAACT ATGCTTTGGTCTTTCTGCAT
mex_AF092566.1_371_520 CTGGCCCTTGAGGTCGCGG CGGTCTTCACCTCGACAC
mex_AF092566.1_50_193 GACGTAGATCGGGTCGAGCT ACGGAAACCTCGGAGAATT
mex_CP000438.1_487178_487357 GGCGTACTGCTGCTTGCTCA TGACGTCGACGTAGATCG
mex_NZ_AAQW01000001.1_461304_461466 CCTGTTCCTGGGTCGAAGCC CTTCGGTCACCGCGGA
erm_EU047809.1_79_229 GTTTATAAGTGGGTAAACCGTGAAT GAAACGAGCTTTAGGTTTGC
gyrB_NC_015663_1455472_1455621 GCCCTTTCAGGACTTTGATACTGG TGTACGGAGACGGAGTTATCG
gyrB_NC_010410_4215_4366 ACACTGACCGATTCATCCTCGTG CTTGAAAGTGCGTTAACAACC
gyrB_NC_005773_4904_5052 CGGAAGCCCACCAAGTGAGTAC CGAAACCAGTTTGTCCTTAGTC
gyrB_NC_016514_5343_5487 ACCAGCTTGTCTTTAGTCTGAGAG CTTTACGACGGGTCATTTCAC
gyrB_NC_016603_2631439_2631616 CATTGGTTTGTTCTGTTTGAGAGGC GATTCATCTTCGTGAATTGTGAC
gyrB_NC_009436_4366_4524 GGACTTTGATACTGGAGGAGTCATA TGTACGGAAACGGAGTTATCG
gyrB_NC_009512_4203_4373 ATGCTGGAGGAGTCGTACGTTT GTCGCGCACACTAATAGATTC
TABLE 13
Plasmid regions can be used for identification purposes and can evidence
horizontal gene transfer.
Probe Coordinates Gene
plasmids_NC_010660_187
035_187205
plasmids_NC_014232_550 parB1, ETEC1392/75_p1018_014, putative ParB plasmid
1_5677 stabilisation protein
plasmids_NC_011838_178 pCAR12_p001, putative ABC transporter
818_178996 subunit, tnpAb, pCAR12_p172, transposase
plasmids_FN554767_1301 EC042_pAA016, site-specific recombinase
7_13190
plasmids_NC_013655_115 ECSF_P1-0138, hypothetical protein
365_115542
plasmids_NC_013951_698
99_70067
plasmids_NC_007635_383 pCoo017, resD, pCoo052, putative resolvase
95_38566
plasmids_NC_009787_179 EcE24377A_C0013, putative methylase
46_18116
plasmids_NC_006671_562 near yfcB, O2R_81, YfcB
59_56438
plasmids_NC_014385_531
51_53310
plasmids_FN649418_5716 ETEC_p948_0010, IS66-family transposase
9_57339
plasmids_NC_005011_862 blaR1, MWP012, bla regulator protein blaRl
0_8785
plasmids_NC_014843_984 yfhA, p3521_p111, YfhA
13_98578
plasmids_NC_008490_516
5_5334
plasmids_NC_015963_147 Entas_4593, integrase catalytic subunit
516_147686
plasmids_NC_007365_100 resD, LH0122, site-specific recombinase
545_100708
plasmids_NC_009838_104 qacEdelta1, APECO1_O1R94, quaternary ammonium
163_104332 compound resistance protein
plasmids_NC_010409_397 pVM01_p034, insertion sequence 2 OrfA protein
68_39935
plasmids_NC_014233_503 ETEC1392/75_p557_00068
37_50492
plasmids_NC_013362_566 ECO26_p2-76, conjugal transfer nickase/helicase TraI
51_56805
TABLE 14
Plasmid arms
Probe Coordinates Binding region 1 Binding region 2
plasmids_NC_010660_187035_187205 GCTGTCACCGTCCAGACGCTGTTGGC TCCGTGCCTTCAAGCGCG
plasmids_NC_014232_5501_5677 GACTCCGCAGAATACGGCACCGTGCGCA GCGTACAGGCCAGTCAGC
plasmids_NC_011838_178818_178996 GCTGTCCTGGCTGCAAGCCTGG CCGAACTGCTGATGGACGT
plasmids_FN554767_13017_13190 GACAGCAGACTCACCGGCTGGTTCCGCT GCAAGATGCTGCTGGCCACACTG
plasmids_NC_013655_115365_115542 GACAGAACAAGTTCCGCTCCGG CACGGATACGCCGCGCAT
plasmids_NC_013951_69899_70067 GAACGTCTGGCGCTGGTCGCCTGCC GCACAGGTGCTGACGTGGT
plasmids_NC_007635_38395_38566 AATCCAGGTCCTGACCGTTCTGTCCGT ACCTCCGTTGAGCTGATGGA
plasmids_NC_009787_17946_18116 GAGGTGGCCAACACCATGTGTGACC GACGCCGGTATATCGGTATCGAGCT
GCT
plasmids_NC_006671_56259_56438 GAAGTGCCGGACTTCTGCAGA GCACGGCCTGATGGAGGCCGC
plasmids_NC_014385_53151_53310 GCTAATCGCATAACAGCTAC CATCACGTAACTTATTGATGATATT
plasmids_FN649418_57169_57339 GCTGCGGTATTCCACGGTCGGCC GCAGGAACGCTGCCTGTGGTC
plasmids_NC_005011_8620_8785 GAATCAATTATCTTCTTCATTATTGAT CTGCGGCTCAACTCAAGCA
plasmids_NC_014843_98413_98578 GTCACACGTCACGCAGTCC GCATTCATGGCGCTGATGGC
plasmids_NC_008490_5165_5334 GTGTTACTCGGTAGAATGCTCGCAAGG ACTAGATGACATATCATGTAAGTT
plasmids_NC_015963_147516_147686 CGGAACTGCCTGCTCGTAT AACGATATAGTCCGTTAT
plasmids_NC_007365_100545_100708 GCTCTCCGACTCCTGGTACGTCAG GCGCGCATTAATGAAGCAC
plasmids_NC_009838_104163_104332 GATGTTGCGATTACTTCGCCAACTATTG GCTGTAATTATGACGACGCCG
plasmids_NC_010409_39768_39935 GCAATACCAGGAAGGAAGTCTTACTG GTCATTGGAGAACAGATGATTGATGT
plasmids_NC_014233_50337_50492 GTATCGCCACAATAACTGCCGGAA AACGATATAGTCCGTTATG
plasmids_NC_013362_56651_56805 GTGAAGCGCATCCGGTCACC ATGGCATAGGCCAGGTCAATAT
TABLE 15
A list of antibiotic resistance genes for which probes can be used to identify,
distinguish and/or sequence
Source Sample ID
CARB
CMY
CTX-M
GES
IMP
KPC
NDM
Other ampC
OXA
PER
SHV
VEB
VIM
ermA
vanA
vanB
mecA
mexA
In some embodiments, the oligonucleic acid probes provided by the invention are molecular inversion probes (MIP). Advantages that the MIP probes described herein offer over PCR include:
1) Multiplexing: there are published studies using 10k+ inversion probes to genotype humans including: http://www.ncbi.nlm.nih.gov/pubmed/17934468 (Porreca et. al.), 55k probes http://www.ncbi.nlm nih gov/pmc/articles/PMC2715272/?tool=pubmed 30k probes http://www.ncbi.nlm.nih.gov/pubmed/19329998 10k probes.
This offers a huge capability to expand panels. First uses might be to capture more rare strains/variants that work poorly with current PCR primers. Later uses might involve genotyping HIV and human loci as well as testing for diseases common in HIV patients—such a test can still be performed in a single tube with minimal per-test increase in reagents cost.
2) Specificity: the probes described herein are less likely to produce off-target products because the two probe arms must bind together. This provides a thermodynamic advantage for on-target binding compared to mis-priming. Furthermore, the exonuclease step will eliminate extension products that occur when only a single probe arm binds.
PCR primers can create long extension products that serve as templates for mis-priming in later rounds. This is particularly a problem when there's lots of background (e.g. human) DNA compared to the target sequence; such as when the exonuclease step didn't remove all of the template and the amplification/barcoding primers misprimed against human DNA. This ends up wasting reads and would have been worse had enrichment for the circularized probes was not being performed. Preventing such reads in a PCR-only system is difficult.
3) Design optimization: the large published datasets provide good training data for a probe picking algorithm. These large datasets can be useful for picking probe sets that will work reliably and with uniform efficiency. Furthermore, we can generate a set of 10k+ probes on a microarray to generate datasets using preferred enzymes. Currently being tested is the entire set of 10k+ probes in a single reaction and then analyzing the read counts to see what made a good probe and what didn't.
Understanding the probe behavior is important for pathogens as it helps to understand the sensitivity and specificity, particularly when considering rare strains or the possibility of previously unknown strains. Pathogenica has thermodynamic models of probe behavior that provide quantitative predictions of how well a probe will work against a target.
4) Simplicity: the probe protocol can be one-tube all the way through, adding reagents until all of the samples are pooled. PCR protocols often require multiple tubes to purify intermediate or final product from the template (e.g., Ampliseq requires 7, PCR+ Nextera likely requires 3+). Also being used are standard reagents (enzymes+oligos) and equipment (thermal cycler).
The following references are incorporated by reference in their entirety: Roberts R R, et al., “Costs attributable to healthcare-acquired infection in hospitalized adults and a comparison of economic methods,” Medical Care, 48(11):1026-1035, November 2010; Scott, R. D., II., “The Direct Medical Costs of Healthcare-Associated Infections in U.S. Hospitals and the Benefits of Prevention,” U.S. Centers for Disease Control and Prevention, March 2009; and Edwards, J. R., et al., National Healthcare Safety Network (NHSN) report: data summary for 2006 through 2008, issued December 2009, American Journal of Infection Control. 37:783-805, December 2009.
It should be understood that for all numerical bounds describing some parameter in this application, such as “about,” “at least,” “less than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description at least 1, 2, 3, 4, or 5 also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.
For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited. Where any conflict exits between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GeneIDs, Unigene IDs, or HomoloGene ID, or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures) are hereby incorporated by reference in their entirety.
Headings used in this application are for convenience only and do not affect the interpretation of this application.
Preferred features of each of the aspects provided by the invention are applicable to all of the other aspects of the invention mutatis mutandis and, without limitation, are exemplified by the dependent claims and also encompass combinations and permutations of individual features (e.g., elements, including numerical ranges and exemplary embodiments) of particular embodiments and aspects of the invention including the working examples. For example, particular experimental parameters exemplified in the working examples can be adapted for use in the claimed invention piecemeal without departing from the invention. For example, for material is that are disclosed, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of elements A, B, and C are disclosed as well as a class of elements D, E, and F and an example of a combination of elements, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, elements of a composition of matter and steps of method of making or using the compositions.
The forgoing aspects of the invention, as recognized by the person having ordinary skill in the art following the teachings of the specification, can be claimed in any combination or permutation to the extent that they are novel and non-obvious over the prior art—thus to the extent an element is described in one or more references known to the person having ordinary skill in the art, they may be excluded from the claimed invention by, inter alia, a negative proviso or disclaimer of the feature or combination of features.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Examples Procedure:
-
- 1) Remove the DxSeq Kit from the −20° C. freezer.
- 2) Remove one Reagent Set Pack from the DxSeq Kit, and place the tubes on ice.
- 3) Remove two blue FrameStrips and matching strip caps from the kit. [The Break-A-Way Plate with primers is not needed at this point in the protocol.]
- 4) Label the FrameStrips and the strip caps #1 and #2 with a permanent marker. [Both the FrameStrips and strip caps should be labeled to avoid cross-contamination during subsequent handling steps.]
- 5) Return the kit to the −20° C. freezer for later use.
- 6) After the components have thawed, pulse-spin any droplets from the cap or sidewalls to the bottom of the tubes using a microcentrifuge.
- 7) Using barrier pipette tips, prepare 75 μL Hybridization Master Mix for 12 samples and 2 controls, as follows:
- a. 22.5 μL 10× Buffer A
- b. 15 μL MIP Probe mixture
- c. 37.5 μl of nuclease-free water
- 8) Using barrier pipette tips, pipette 5 μL of Hybridization Master Mix into wells A-G of two blue FrameStrip PCR 8-strips (n=14 wells). [Do not pipette Hybridization Master Mix into wells H: these are reserved for negative controls.]
- 9) Being very careful not to cross-contaminate the wells, add 10 μL of each DNA sample to the A-F wells of the two FrameStrips (n=12 wells). [Do not pipette your DNA samples into the G & H wells: these four wells are reserved for control reactions.]
- 10) Add 10 μL of nuclease-free water to the G wells (n=2 wells). These will serve as the “no target DNA” negative controls.
- 11) Add 13.5 of nuclease-free water and 1.5 of 10× Buffer A to the H wells (n=2 wells). These will serve as the “no probe” negative controls.
- 12) Seal the two FrameStrips with the flat strip caps.
- 13) Vortex the sealed FrameStrips briefly to mix the contents; and then pulse-spin down the contents in a microcentrifuge with a rotor that accommodates 8-well strip PCR tubes.
- 14) Enter the following program into a thermocycler, using the heated lid option.
a. 94° C., 10 min Hybridization
b. Ramp to 60° C., 0.1° C./sec
c. 60° C., 10 min
d. 60° C. hold
e. 60° C., 10 min Extension
f. 15° C. hold
g. 94° C., 2 min Exonuclease cleanup
h. 37° C. hold
i. 37° C., 30 min
j. 94° C., 15 min
k. 4° C. hold
-
- 15) Place the sealed FrameStrips in the thermocycler; and begin the hybridization portion of the MIP Program.
- 16) While the hybridization is underway, prepare the Polymerase/Ligase Master Mix on ice:
- a. 5 μL Polymerase
- b. 5 μL 10× Buffer A
- c. 1 μL Ligase
- d. 1.25 μL dNTPs
- e. 37.75 μL nuclease-free water
- 17) When the hybridization reaction reaches the 60° C. hold step (approximately 26 minutes into the program), add 2 μL of the Polymerase/Ligase Master Mix to every well (n=16 wells).
- 18) Reseal the FrameStrips with the same strip caps as before and mix. [Special care needs to be taken not to cross-contaminate the samples.]
- 19) Advance the thermocycler to the next step in the MIP Program (60° C. for 10 min).
- 20) When the thermocycler reaches the 15° C. hold step, advance the thermocycler to the next step (94° C. for 2 min) in the MIP Program.
- 21) When the thermocycler reaches the 37° C. hold step, immediately add 1 μL of Exonuclease to each sample.
- 22) Reseal the FrameStrips with the same strip caps as before and mix. [Special care needs to be taken not to cross-contaminate the samples.]
- 23) Advance the thermocycler to the next step (37° C. for 30 min) in the MIP Program.
- 24) While the reactions are incubating at 37° C., prepare the amplification mix:
- a. (components of the PCR reaction)
- 25) Remove the Purple Break-A-Way 96 Well Plate containing PCR primers from the −20° C. freezer. Break off three columns from the left side of the plate.
- 26) Return the unused portion of the Break-A-Way 96 Well Plate to the freezer (before the primers thaw).
- 27) When the thermocycler reaches the 4° C. hold, add 2.5 μL of tube-specific barcoding primer and 29.5 μL of amplification mix.
- 28) Begin the PCR Amplification Program on the thermocycler:
- a. 94° C., 3 min
- b. 30 cycles of:
- i) 94° C., 15 sec
- ii) 60° C., 15 sec
- iii) 72° C., 30 sec
- c. 72° C., 4 min
- d. 4° C. hold
- 29) Purify the PCR amplicons using AMPure beads (Beckman Coulter).
- 30) Proceed to the IonTorrent Template preparation workflow.
Pathogenica Software installed on the Ion Torrent PGM reports the results.
